Hormone receptor-like in 46



Hr46 mRNA is detected during midembryogenesis (9-18) hours (Koelle, 1992). Hr46 is induced following germ band retraction [Images] and can be detected until cuticle deposition late in embryogenesis. Hr46 is widely expressed during mid-embryogenesis in tissues that include the gut, salivary gland, ventral nerve cord and epidermis. The embryonic central nervous system contains little, if any DHR protein (Lam, 1997).

The Drosophila nuclear receptors DHR3 and βFTZ-F1 control overlapping developmental responses in late embryos

Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, βFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, βFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. This study turned to embryonic development to address these two issues. It was shown that mid-embryonic expression of DHR3 and βFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 andβFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and βFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of βFTZ-F1. DHR3 and βFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and βFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade (Ruaud, 2010).

The regulatory interactions between DHR3, αFTZ-F1 and E74A/E75A that are described in this study in embryos are indistinguishable from those seen in prepupae. First, DHR3 expression in embryos is dependent on 20E signaling. Second, DHR3 mutants display reduced levels of αFTZ-F1, E74A and E75A expression at both stages in the life cycle, and αFTZ-F1 mutants have reduced levels of E74A mRNA and no detectable E75A expression. Taken together with studies that show that ectopic αFTZ-F1 is sufficient to drive maximal expression of E74A and E75A, these results indicate that DHR3 exerts its effect on these genes through its induction of αFTZ-F1 in embryos. Third, a loss of DHR3 function during embryogenesis does not eliminate αFTZ-F1 expression. This is probably due to other upstream factors that contribute to this response. One candidate for this function is the DHR4 nuclear receptor, which is coexpressed with DHR3 in both embryos and prepupae. DHR4 mutants have no effect on DHR3 expression, but display significantly reduced levels of αFTZ-F1 mRNA in prepupae. These mutants, however, have no effect on embryonic development, suggesting that DHR4 does not play a major role in αFTZ-F1 induction at this early stage in the life cycle (Ruaud, 2010).

The late larval pulse of 20E both directly and indirectly induces DHR3 and represses αFTZ-F1. Taken together with the inductive effect of DHR3 on αFTZ-F1 expression, this regulation ensures that the peak of αFTZ-F1 expression will be delayed until the proper time during development. The observation that the embryonic 20E pulse, at ~8 hours AEL, immediately precedes DHR3 expression suggests that similar regulatory interactions are acting in embryos. However, unlike prepupae, there is no known hormone peak in late embryos that could account for the coordinated induction of E74A and E75A mRNA at this time, as is known to occur in late prepupae. It is possible that these transcripts are fully dependent on trans-acting factors such as αFTZ-F1 for their expression in embryos. Alternatively, these 20E primary-response genes might be induced by a novel temporal signal that remains to be identified (Ruaud, 2010).

It is interesting to note that a similar temporal profile of DHR3, αFTZ-F1 and E74A/E75A expression is also seen in larvae. A burst of DHR3 expression in mid-second instar larvae immediately follows the peak in the 20E titer and precedes the transient expression of αFTZ-F1, which is followed by co-expression of E74A and E75A at the end of the instar. Curiously, E75A, but not E74A, is expressed at an earlier time as well, in apparent synchrony with the 20E pulse, recapitulating the timing seen in embryos. It is thus likely that a common set of regulatory interactions function in both embryos and larvae to dictate the precise timing of these expression patterns at each stage in the life cycle, prior to the third instar. Moreover, the observation that EcR, E75A and αFTZ-F1 mutants display defects in larval molting indicates that their expression is essential for proper progression through these stages in development (Ruaud, 2010).

DHR3 and αFTZ-F1 null mutations lead to fully penetrant embryonic lethality, with relatively minor and partially penetrant phenotypes reported in DHR3 mutant embryos and no phenotypic description of αFTZ-F1 mutant embryos. The studies described in this paper define both common and unique functions for these two nuclear receptors during embryogenesis. DHR3 and αFTZ-F1 null mutants both display a highly penetrant defect in air filling of the tracheal tree. In addition to this common function, αFTZ-F1 is required for the proper differentiation of the denticles in the ventral cuticle and DHR3 is required for VNC condensation. Both DHR3 and αFTZ-F1 mutants display apparently normal muscle movements at the end of embryogenesis, indicating that only some developmental responses are blocked at this stage. These processes of cuticle differentiation, tracheal air filling, muscular movements and VNC condensation represent the major developmental events that can be described in late embryos. Defects in three of these four pathways thus define a central role for DHR3 and αFTZ-F1 in late embryonic development. In addition, unlike prepupae, in which DHR3 and αFTZ-F1 mutants have essentially identical phenotypes, these studies establish independent functions for these two nuclear receptors during development. Together with the previously identified early embryonic roles of the 20E receptor EcR in dorsal closure, head involution and midgut morphogenesis, these data indicate that each step of the 20E-induced transcriptional cascade controls sequential developmental programs during embryogenesis. Moreover, the observation that this transcriptional cascade is also required for larval molting suggests that it represents a stereotypic 20E response that is required for progression through each major transition in the life cycle (Ruaud, 2010).

Ectopic expression of wild-type αFTZ-F1 is sufficient to rescue the lethality of αFTZ-F1 mutants, but has no effect on the viability of DHR3 mutants, indicating that DHR3 exerts essential functions independently of its downstream partner. The causes of lethality in DHR3 and αFTZ-F1 mutant embryos, however, remain unclear. Strong loss-of-function mutations in the signal peptide peptidase (Spp) gene result in tracheal air-filling defects; however, Spp mutant embryos hatch normally and die as first or second instar larvae. Similarly, embryos with severe defects in VNC condensation can hatch into first instar larvae and survive to later stages of development. These results indicate that the lethality of DHR3 and αFTZ-F1 mutant embryos cannot be directly attributed to defects in these pathways. Rather, DHR3 and αFTZ-F1 may participate in a developmental checkpoint necessary to trigger the last steps of embryogenesis required for hatching and survival (Ruaud, 2010).

The microarray study revealed that a number of 20E-responsive genes are misregulated in DHR3 mutants, consistent with studies in prepupae that indicate a crucial role for DHR3 in 20E signaling. The microarray analysis also identified several genes that are involved in chitin metabolism and protein secretion, which could account for the defects in tracheal gas filling seen in DHR3 mutants. These included the chitinase genes Idgf5 (-8.6-fold) and kkv (+2.4-fold), the CBP Cht12 (+2.6-fold) and the COPII coat subunit sec13 (+2.5-fold). This study also identified a number of genes that play a role in axon guidance. Interestingly, most of these genes have dose-dependent effects, whereby either reduced or increased expression can disrupt nervous system development. Failure of DHR3 mutant embryos to express these genes at normal levels could thus contribute to the PNS defects (Ruaud, 2010).

Northern blot hybridization studies to examine the effects of DHR3 and αFTZ-F1 mutants on selected DHR3-regulated genes confirm and extend phenotypic studies of these mutants. Some genes, such as retn, E93 and kkv, display similar transcriptional responses in DHR3 and αFTZ-F1 mutants, whereas E74A and E75A are more significantly affected in αFTZ-F1 mutants and Idgf5 is selectively reduced in DHR3 mutants. These transcriptional effects support phenotypic studies and provide further evidence that DHR3 and αFTZ-F1 exert common and independent regulatory roles during embryogenesis. This conclusion is consistent with experimental and theoretical studies of gene regulatory networks, which indicate that transcriptional cascades provide an effective means of amplifying signals and integrating multiple cues to provide specificity in biological responses. Transcriptional cascades can also direct temporal programs of successive gene expression, as observed in the formation of flagella in Escherichia coli and the specification of anteroposterior patterning in the Drosophila embryo. In addition, the DHR3-αFTZ-F1 transcriptional cascade involves nuclear receptors that could potentially act as ligand-regulated transcription factors, introducing an additional level of control by small lipophilic compounds. These observations support the proposal that the sequential expression of DHR3 and αFTZ-F1 at multiple stages of development can specify successive biological programs that promote appropriate progression through the life cycle. By combining insect endocrinology with the predictive power of genetics, the 20E-triggered transcriptional cascades in Drosophila provide an ideal context to define how a repeated systemic signal can be refined into precise stage-specific temporal responses during development (Ruaud, 2010).

DHR3 is required for VNC condensation, a terminal step in embryonic nervous system morphogenesis that is dependent on nervous system activity, glial cell function and apoptosis. In addition, previous studies have identified roles for DHR3 in PNS development. Interestingly, these functions, which are specific for DHR3 and are not shared with its direct target, αFTZ-F1, parallel the role of the mammalian DHR3 homolog RORα in brain development. RORα was initially identified as the gene associated with the spontaneous staggerer mutation in mice, which display ataxia associated with cerebellum developmental defects and degeneration. The cerebellum in staggerer mutants is dramatically smaller than in controls, containing fewer of the two major cell types: granule cells and Purkinje cells. Further investigation showed that this phenotype arises primarily from reduced expression in Purkinje cells of Sonic hedgehog (Shh), a mitogenic signal for granule cells. These data support the hypothesis that there is an evolutionarily conserved role for the ROR/DHR3 family of nuclear receptors in nervous system development and suggest that further functional studies of DHR3 may provide new insights into its ancestral functions in this pathway (Ruaud, 2010).

Larval and pupal stages

Hr46 mRNA is detected during the first two larval stages or instars and at the end of the third larval instar (Koelle, 1992).

The temporal developmental profile for Hr46 expression closely parallels that for the ecdysone titer and for the ecdysone-inducible E75 and E74 Drosophila early genes. The structural similarity to a Manduca early gene and the expression similarities to Drosophila early genes suggest that the Hr46 gene may also belong to the early gene class (Koelle, 1992).

Hr46 is expressed in the larval and imaginal cells of the salivary gland, as well as the fat bodies, Malpighian tubules and imaginal discs of 0-2-hour prepupae. Although expression is clearly evident in the peripodial membranes that surround imaginal discs, Hr46 protein is also present in the disc epithelial monolayer. In addition, Hr46 is found in the midgut, garland cells, trachea, ring gland, hemocytes, lymph gland and pericardial cells of early prepupae. Only low levels of Hr46 protein are present in the 2-hour prepupal central nervous system and no protein can be detected in the proventriculus (Lam, 1997).

Many nuclear receptor genes are localised in the polytene chromosome puffs of the salivary gland previously classified as intermoult, early or early-late puff loci. Early-late puffs include Hr46/DHR3, DHR-39 and E78B. These puffs appear after those of Ecdysone receptor, E74A and E75A, but prior to betaFTZ-F1 and the late puffs. Transcripts from certain early-late puffs are induced in parallel with the early transcripts and are thus hierarchically equivalent. Hr46 is not induced in the absence of ecdysone. The induction of Hr46 does not require protein synthesis and is a primary response to hormone. Particularly striking are the mirror image of Hr46 and Ecdysone receptor in vivo. Hr46 does not become activated while EcR levels are high. This suggests that the best candidate for EcR repression is a gene whose regulation is similar to that of Hr46, and this cross-talk may continue later in the prepupal period (Huet, 1995).

Pulses of the steroid hormone 20-hydroxyecdysone (20E) trigger the larval-to-adult metamorphosis of Drosophila by reprogramming gene expression throughout the organism. 20E directly induces a small set of early regulatory genes that repress their own expression and induce a large set of late secondary-response genes. Two members of the Drosophila nuclear hormone receptor superfamily, Hr46/DHR3 and DHR39, are rapidly induced by 20E, in parallel with the early regulatory genes. Both genes also require protein synthesis at high 20E concentrations for their maximal induction by the hormone. DHR39 is induced in mid third instar larvae and expressed throughout most of third instar larval and prepupal development, while Hr46 is briefly expressed in late third instar larvae and early prepupae. The 20E-induction and temporal patterns of Hr46 and DHR39 transcription strongly suggest that these genes function together with the early regulatory genes to coordinate the complex gene networks that direct the early stages of Drosophila metamorphosis (Horner, 1995).

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA+ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

Description of wild-type development is a first step in understanding metamorphosis from a global perspective. However, it is of interest to understand the composition of the genetic hierarchy that leads to metamorphosis. To test whether new targets of transcription factors could be identified in the ecdysone genetic hierarchy, the ecdysone-induced nuclear receptor DHR3 was prematurely expressed at >18 hours BPF. DHR3 is responsible for the coordination of part of the transcriptional program controlling metamorphosis and can act as either a repressor or an activator of transcription, depending on the target gene. DHR3 can induce betaFTZ-F1, a nuclear receptor that is active during midprepupal development and is responsible for the difference in the genetic response to ecdysone between the late larval and prepupal ecdysone pulses. betaFTZ-F1 induction is confirmed by the microarray results. Several other genes are induced by DHR3 when it is expressed at >18 hours BPF. One of these is represented by a novel EST (LD24139) that is induced from 3 to 9 hours APF during wild-type development. ESTs representing 12 other DHR3-induced genes that have less than threefold induction at 3 to 9 hours APF are listed at DHR3-regulated genes. Some of these additional genes may not normally be DHR3 targets or may be induced by DHR3 at other stages during development (White, 1999).

DHR3 has been shown to inhibit the induction of ecdysone-inducible genes, and with E75B it can act as a repressor of the betaFTZ-F1 gene. DHR3 is expressed before the ecdysone-inducible genes are up-regulated but is still capable of repressing genes. Four out of seven such genes belong to the cytochrome P450 (CYP) class of genes. Three of these CYP genes are normally repressed during the late larval pulse, and this repression begins before DHR3 induction occurs (approximately 4 hours BPF). Thus, DHR3 cannot be solely responsible for their repression, although it may contribute to it. One function of cytochrome P450 molecules is hydroxylation of steroids; the depletion of transcripts of the CYP genes may provide a mechanism by which production of the biologically active form of ecdysone (20-hydroxyecdysone) is stifled at PF. Regulation of these CYP genes within the ecdysone hierarchy further suggests that they may have a role in controlling the ecdysone genetic cascade (White, 1999).

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).

The HR46 gene (also known as DHR3) encodes a nuclear receptor and is an essential gene known to be important for the ecdysone cascade. Large clones of loss of function alleles result in wing (folded and curved) and notum defects (rough short bristles and pale pigmentation). The expression of this gene increased 250 fold from 24 to 32 hr and then decreased 4.3 fold from 32 to 40 hr. Moderate sized wing clones of cells lacking HR46 were examined, but no clear cut phenotype was seen. In pupal wing clones examined a couple of hours after hair formation mutant hairs appeared somewhat thicker but this alteration was transient (Ren, 2005).

The Eip78CD gene encodes a related nuclear receptor. The expression of this non-essential gene increased 3 fold from 24 to 32 hr followed by a three fold drop from 32 to 40 hr (but the differences were not significant) suggesting it might be functionally redundant with HR46. To test this hypothesis Eip78CD mutants, which also contained HR46 mutant clones, were examined. No mutant phenotypes were seen in the clones, suggesting either that there is an alternative redundant gene or that HR46 is not essential for hair morphogenesis. Since the level of HR46 expression fell dramatically between 32 and 40 hrs it seemed possible that declining HR46 expression could be important for hair development. To test this the overexpression of HR46 from a transgene containing a hs promoter was induced. This resulted in a dramatic loss of hair formation leading to wings with extensive bald regions. The strongest phenotype was seen when the transgene was induced by heat shocking 6-8 hrs prior to the time of hair initiation. The phenotype was dose sensitive and directly related to the number of transgenes and length and temperature of transgene induction (Ren, 2005).

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).

Effects of Mutation or Deletion

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. A reverse genetics approach was used to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo (Carney, 1997).

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. A heat-inducible DHR3 rescue construct and clonal analysis were used to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, but no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. About half of the DHR3 mutants rescued to the third larval instar display defects in their tracheal system. The taenidial folds of the tracheal cuticle can be severely distorted. In more severe cases, this can lead to collapse of the tracheal cuticle and obstruction of the lumen, followed by necrosis. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-F1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. Clonal analysis provides support for a role for DHR3 in larval muscle development. The larval muscles of DHR3 mutants may provide a defective template for adult muscle formation, leading to a held out wing phenotype. DHR3 is inhibitory to late third instar larval gene expression, including BR-C, E74A and E75A, while DHR3 acts positively on later gene expression in mid-prepupae. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis (Lam, 1999).

The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila

S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, a Drosophila-based, sensitized, gain-of-function genetic screen was used. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. This study demonstrates that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, the ligand-binding domain (LBD) of DHR3 was shown to be essential for mediating this response. Consistent with these findings, an endogenous DHR3 isoform was identified that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis (Montagne, 2009).

By using Drosophila genetics and a gain-of-function strategy, the NR, DHR3, was identified as an enhancer of a dS6K-regulated growth phenotype. This effect can be mediated by an isoform of DHR3 lacking the DBD. Moreover, using a revertant screening strategy, LBD-specific DHR3 mutants were generated, and it was demonstrated that the LBD of DHR3 is necessary to maintain normal growth and dS6K activity. In contrast to the role DHR3 plays in transcriptional regulation affecting the onset of metamorphosis, these studies indicate that it also plays a role in regulating cell-autonomous growth. These effects are most likely mediated through dS6K, as the ability of ectopically expressed DHR3-RS to drive growth in the dorsal wing blade is blunted in Drosophila deficient for dS6K. Consistent with these findings, it has been demonstrated that dS6K also controls cell growth in a cell-autonomous manner. However, the effect on cell size is more pronounced in dS6K mutants than in the DHR3-mutant clones described in this study. This may reflect the fact that dS6K activity is blunted, but not abolished, in DHR3 LBD-mutant larvae. Compatible with this hypothesis, it was found that in a dS6K P-element-induced mutant (P{PZ}S6K[07084]) no dS6K protein could be detected; however, this mutation induced a much less severe phenotype as compared with the dS6Kl-1 null mutation. In homozygous DHR3 mutant eyes both the size and the number of ommatidia were decreased, whereas in dS6K mutant flies the size reduction of the eye was only due to a decrease in ommatidia size but not number. This difference might be attributed to the experimental settings. In the current study, DHR3 mutant eyes were generated by mitotic recombination in a heterozygous Minute background, whose developmental delay is less than two days. In contrast, the size and number of ommatidia in dS6K mutant eyes were measured in homozygous mutant flies that exhibit a five-day delay at eclosion. The longer time for the latter to emerge as adults allows additional cell divisions to proceed, leading to a higher number of ommatidia (Montagne, 2009).

Previous studies demonstrated that DHR3 participates in a hierarchal regulatory circuit in response to ecdysone signaling, but also acts in a negative feedback loop to repress ecdysone receptor-mediated signaling. Prothoracic gland production of ecdysone is mediated by the brain neuropeptide prothoracicotropic hormone (PTTH). Recent studies in Drosophila have shown that genetic ablation of PTTH-producing neurons induces a delay in larval development and results in larger adult flies as a direct consequence of reduced levels of ecdysone. Interestingly, in the tobacco hornworm, Manduca sexta, PTTH-induced ecdysone production is paralleled by the phosphorylation of the Manduca orthologue of Drosophila ribosomal protein S6. Moreover, this process is sensitive to rapamycin, and a burst of dS6K activity is observed at early pupation. As the body size of the adult fly appears to be determined by growth regulators, including dS6K, as well as by hormones that control the timing of developmental windows, such as PTTH, the results suggest that the DHR3/dS6K regulatory module acts to integrate these two processes (Montagne, 2009).

These studies supports the existence of a novel DHR3 polypeptide devoid of a DBD, DHR3-PS. Nonetheless, although DHR3-PS is sufficient to potentiate a dS6K-dependent growth phenotype, it is not possible to exclude that the other DBD-containing DHR3 isoforms also contribute to dS6K activation. In general, DHR3, like other NRs, is a transcription factor composed of four elements: a modulator domain, the DBD, the hinge region, and the LBD. The DBD of NRs typically consists of two zinc fingers, with the first being critical for conferring DNA-binding specificity. Like DHR3-PS, NRs lacking a DBD have been previously reported. Notably, in Drosophila, the NR E75B, a DHR3 partner, lacks one of the 2 zinc fingers that is required to form a functional DBD. However, E75B, through its ability to interact with DHR3, modulates DHR3 transcriptional activity in a gas-responsive manner (Reinking, 2005). Like the putative DHR3-PS, the NR short heterodimer partner (SHP) in mammals is also devoid of DBD, but, as with E75B, it interacts with other NRs to modulate their transcriptional activity. It is unlikely that DHR3-PS behaves as a dominant-interfering effector of full-length DHR3 as ectopic DHR3-PS expression induces growth, whereas DHR3-RNAi inhibits growth. However, DHR3 also heterodimerizes with two NRs: E75 and the ecdysone receptor. Thus, in the case of E75, ectopically expressed DHR3-PS may act to decrease the levels of free E75, leaving full-length DHR3 free to increase the transcription of target genes. In contrast, DHR3-PS binding to the ecdysone receptor could counteract the negative growth regulation mediated by ecdysone signaling. However, it should be noted that the negative effects of ecdysone are humoral and mediated by dFOXO-inactivation within the fat body, whereas, as this study has shown, DHR3 regulates growth in a cell-autonomous manner. Moreover, dFOXO subcellular distribution was not altered in DHR3 mutant clones in third instar wing imaginal discs, indicating that the DHR3 cell-autonomous effect on cell growth is not mediated by the PKB/dFOXO signaling (Montagne, 2009).

In contrast to acting as a dominant-interfering isoform, the results presented in this study also suggest that DHR3 activates dS6K through a non-genomic mechanism, an effect of NRs that does not require the DBD function. Such a model is supported by NR responses whose kinetics are too rapid to be explained by de novo transcription and translation of a gene product. Indeed, nongenomic effects typically occur within minutes following addition of the cognate ligand and are resistant to transcriptional inhibitors. In the case of DHR3, it is experimentally difficult to address this question as the ligand for DHR3 is unknown, and a genetic endpoint is being scored for, resulting from events induced much earlier in larval development. It has been demonstrated that vitamin D3 and all-trans-retinoic acid both induce activation of S6K1 within minutes of administration to cells. Moreover, in the case of vitamin D3, it was shown that these effects were mediated through protein phosphatases PP1 and PP2A in a vitamin D3 receptor (VDR)-dependent manner. VDR appears to directly interact with the catalytic subunits of PPI and PP2A, and vitamin D3 acts to disrupt this interaction and enhance an interaction between VDR and S6K1, stabilizing S6K1 in its phosphorylated active state. However, depleting DHR3 levels by RNA interference blunts both dS6K T398 and d4E-BP T37/T46 phosphorylation, suggesting that DHR3 acts upstream or at the level of dTORC1. Identification of potential partners for DHR3-PS may be useful in determining, at the molecular level, the mechanism by which DHR3 controls cell growth and dS6K activity (Montagne, 2009).

The data further support the notion that a ligand exists for DHR3, and that the ligand is required for many of the pleiotropic activities of DHR3. Those NRs that bind steroid hormones are, in general, high-affinity receptors, whereas the low-affinity NRs bind ligands that are present in high concentration, such as dietary nutrients. The observation that an NR, generated by fusing the DHR3 LBD with the DBD of Gal4, is transcriptionally active in a number of specific embryonic and larval tissues suggests that such a ligand is widely present. Given the role of dTOR/dS6K as a nutritional effector, it is interesting to note that the chimeric DHR3/Gal4 NR is active in organs that provide basal nutrients, in particular, in a group of cells of the larval midgut, which are essential for the transfer of nutrients to the hemolymph. Importantly, the mammalian orthologues to DHR3 and its partner E75 are retinoid-related orphan receptor (ROR)α and Rev-erb (NR1D)α, respectively. As in Drosophila, the NR1D subgroup functions as dominant transcriptional silencers by inhibiting transactivation mediated by RORα. Interestingly, it was recently reported that RORα-deficient mice, like S6K1-deficient mice, exhibit reduced fat-pad mass, smaller adipocytes, and resistance to diet-induced obesity. Moreover, in solving the X-ray structure of the RORα LBD, it was revealed that cholesterol was bound in the ligand-binding pocket. While the Drosophila NR, DHR96, has recently been shown to bind cholesterol thereby modulating cholesterol homeostasis, this does not exclude the possibility that DHR3 could also bind cholesterol. However, the predicted models of the structure of DHR3 indicate that the size of the ligand-binding pocket is smaller than those of either RORα or RORβ. Given the role of the mTOR/S6K1 nutrient-responsive pathway in mammals, it raises the possibility that DHR3 is a low-affinity receptor for an abundant nutrient ligand. Identification of this specific ligand constitutes the next issue to investigate (Montagne, 2009).

Blimp-1/PRDM1 and Hr3/RORbeta specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway

During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORbeta activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. This study shows that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORbeta promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, this study identified a novel interaction between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision (Bunker, 2023).


Akashi, M. and Takumi, T. (2005). The orphan nuclear receptor RORalpha regulates circadian transcription of the mammalian core-clock Bmal1. Nat. Struct. Mol. Biol. 12: 441-44. 15821743

Beckstead, R., et al. (2001). Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit FTZ-F1-dependent transcription. Molec. Cell 7: 753-765. 11336699

Bunker, J., Bashir, M., Bailey, S., Boodram, P., Perry, A., Delaney, R., Tsachaki, M., Sprecher, S. G., Nelson, E., Call, G. B. and Rister, J. (2023). Blimp-1/PRDM1 and Hr3/RORbeta specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway. Front Cell Dev Biol 11: 1058961. PubMed ID: 36960411

Cáceres, et al. (2011). Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75. Genes Dev. 25(14): 1476-85. PubMed Citation: 21715559

Carlberg, C., and Wiesenberg, I. (1995). The orphan receptor family RZR/ROR, melatonin and 5-lipoxygenase: an unexpected relationship. J Pineal Res 18 (4): 171-178. PubMed Citation: 8531047

Carney, G. E., et al. (1997). DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis. Proc. Natl. Acad. Sci. 94(22): 12024-12029. PubMed Citation: 9342356

Colombani, J., et al. (2005). Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science 310: 667-670. PubMed Citation: 16179433

Duez, H., Staels, B. (2009a). Rev-erb-α: an integrator of circadian rhythms and metabolism. J. Appl. Physiol. 107: 1972-1980. PubMed Citation: 19696364

Duez, H., et al. (2009b). Inhibition of adipocyte differentiation by RORα. FEBS Lett 583: 2031-2036. PubMed Citation: 19450581

Dussault, I. and Giguere, V. (1997). Differential regulation of the N-myc proto-oncogene by ROR alpha and RVR, two orphan members of the superfamily of nuclear hormone receptors. Mol Cell Biol 17 (4): 1860-1867. PubMed Citation: 9121434

Forman, B. M., et al. (1994). Cross-talk among ROR alpha 1 and the Rev-erb family of orphan nuclear receptors. Mol Endocrinol 8 (9): 1253-1261. PubMed Citation: 7838158

Frasch, M., Chen, K. and Lufkin, T. (1995). Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila. Development 121: 957-974. PubMed Citation: 7743939

Greiner, E. F., et al. (1996). Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor. Proc. Natl. Acad. Sci. 93 (19): 10105-10110. PubMed Citation: 8816759

Giguere, V., et al. (1994). Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. Genes Dev 8 (5): 538-553. PubMed Citation: 7926749

Giguere, V., McBroom, L. D., and Flock, G. (1995). Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor. Mol Cell Biol 15 (5): 2517-2526. PubMed Citation: 7739535

Gold, D. A., et al. (2003). RORalpha coordinates reciprocal signaling in cerebellar development through Sonic hedgehog and calcium-dependent pathways. Neuron 40: 1119-1131. 14687547

Hamilton, B. A., et al. (1996). Disruption of the nuclear hormone receptor RORalpha in staggerer mice. Nature 379: 736-739. 8602221

Hayes, G. D., Frand, A. R. and Ruvkun, G. (2006). The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25. Development 133(23): 4631-41. Medline abstract: 17065234

Hirose, T., Smith, R. J., and Jetten, A. M. (1994). ROR gamma: the third member of ROR/RZR orphan receptor subfamily that is highly expressed in skeletal muscle. Biochem Biophys Res Commun 205 (3): 1976-1983

Horner, M. A., Chen, T., and Thummel, C. S. (1995). Ecdysteroid regulation and DNA binding properties of Drosophila nuclear hormone receptor superfamily members. Dev Biol 168 (2): 490-502

Huet, F., Ruiz, C., and Richards, G. (1995). Sequential gene activation by ecdysone in Drosophila melanogaster: the hierarchical equivalence of early and early late genes. Development 121 (4): 1195-1204

Jindra, M., Sehnal, F., and Riddiford, L. M. (1994). Isolation and developmental expression of the ecdysteroid-induced GHR3 gene of the wax moth Galleria mellonella. Insect Biochem Mol Biol 24 (8): 763-773

Kageyama, Y., et al. (1997). Temporal regulation of the mid-prepupal gene FTZ-F1: DHR3 early late gene product is one of the plural positive regulators. Genes Cells 2(9): 559-69

Kallen, J., et al. (2004). Crystal structure of the human RORalpha ligand binding domain in complex with cholesterol sulfate at 2.2 Å. J. Biol. Chem. 279(14): 14033-8. 14722075

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

Koelle, M. R., Segraves, W. A. and Hogness, D. S. (1992). DHR3: a Drosophila steroid receptor homolog. Proc. Natl. Acad. Sci. 89 (13): 6167-6171

Koibuchi, N. and Chin, W. W. (1998). ROR alpha gene expression in the perinatal rat cerebellum: ontogeny and thyroid hormone regulation. Endocrinology 139(5): 2335-2341. 9564842

Koibuchi, N., et al. (1999). ROR alpha augments thyroid hormone receptor-mediated transcriptional activation. Endocrinology 140(3): 1356-1364. 10067863

Kostrouch, Z., Kostrouchova, M., and Rall, J. E. (1995). Steroid/thyroid hormone receptor genes in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 92 (1): 156-159

Kostrouchova, M., et al. (1998). CHR3: a Caenorhabditis elegans orphan nuclear hormone receptor required for proper epidermal development and molting. Development 125: 1617-1626

Kostrouchova, M., et al. (2001). Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. 98: 7360-7365. 11416209

Lam, G. T., Jiang, C., and Thummel, C. S. (1997). Coordination of larval and prepupal gene expression by the DHR3 orphan receptor during Drosophila metamorphosis. Development 124 (9): 1757-1769

Lam, G., et al. (1999). DHR3 Is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis. Dev. Biol. 212(1): 204-216

Lan, Q., Wu, Z. and Riddiford, L. M. (1997). Regulation of the ecdysone receptor, USP, E75 and MHR3 mRNAs by 20-hydroxyecdysone in the GV1 cell line of the tobacco hornworm, Manduca sexta. Insect Mol. Biol. 6 (1): 3-10

Lan, Q., et al. (1999). Activation of a delayed-early gene encoding MHR3 by the ecdysone receptor heterodimer EcR-B1-USP-1 but not by EcR-B1-USP-2. Mol. Cell. Biol. 19(7): 4897-4906. 10373539

Lau, P., et al. (2005). RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR. J. Biol. Chem. 279(35): 36828-40. 15199055

Mangelsdorf, D. J., et al. (1995). The nuclear receptor superfamily: the second decade. Cell 83: 835-839

Marvin, K. A., et al. (2009). Nuclear receptors homo sapiens Rev-erbbeta and Drosophila melanogaster E75 are thiolate-ligated heme proteins which undergo redox-mediated ligand switching and bind CO and NO. Biochemistry 48(29): 7056-71. PubMed Citation: 19405475

Matsui, T. (1996). Differential activation of the murine laminin B1 gene promoter by RAR alpha, ROR alpha, and AP-1. Biochem Biophys Res Commun 220 (2): 405-410

McBroom, L. D., Flock, G. and Giguere, V. (1995). The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions. Mol Cell Biol 15 (2): 796-808

Miki, N., Ikuta, M. and Matsui, T. (2004). Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1. J. Biol. Chem. 279(15): 15025-31. 14742449

Montagne, J., et al. (2009). The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. PLoS Genet. 6: e1000937. PubMed Citation: 20463884

Nakagawa, S., Watanabe, M. and Inoue, Y. (1997). Prominent expression of nuclear hormone receptor ROR alpha in Purkinje cells from early development. Neurosci. Res. 28(2): 177-184. 9220474

Nakajima, Y., et al. (2004). Bidirectional role of orphan nuclear receptor RORalpha in clock gene transcriptions demonstrated by a novel reporter assay system. FEBS Lett. 565(1-3): 122-6. 15135064

Niederreither, K., et al. (1997). Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development. Mech. Dev. 62 (1): 67-78

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

Palli, S. R., Hiruma, K., and Riddiford, L. M. (1992). An ecdysteroid-inducible Manduca gene similar to the Drosophila DHR3 gene, a member of the steroid hormone receptor superfamily. Dev Biol 150 (2): 306-318

Palli, S. R., et al. (1996). Cloning and developmental expression of Choristoneura hormone receptor 3, an ecdysone-inducible gene and a member of the steroid hormone receptor superfamily. Insect Biochem Mol Biol 26 (5): 485-499

Palli, S. R., Ladd, T. R., and Retnakaran, A. (1997). Cloning and characterization of a new isoform of Choristoneura hormone receptor 3 from the spruce budworm. Arch Insect Biochem Physiol 35 (1-2): 33-45

Pardee, K. I., et al. (2009). The structural basis of gas-responsive transcription by the human nuclear hormone receptor REV-ERBα. PLoS Biol 7: e1000043. PubMed Citation: 19243223

Parvy, J. P., Wang, P., Garrido, D., Maria, A., Blais, C., Poidevin, M. and Montagne, J. (2014). Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster. Development 141(20):3955-65. PubMed ID: 25252945

Preitner, N., et al. (2002). The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110(2): 251-60. 12150932

Reinking, J., et al. (2005). The Drosophila nuclear receptor E75 contains heme and is gas responsive. Cell 122: 195-207. 16051145

Ren, N., Zhu, C., Lee, H. and Adler, P. N. (2005). Gene expression during Drosophila wing morphogenesis and differentiation. Genetics 171(2): 625-38. 15998724

Rewitz, K. F., Yamanaka, N., Gilbert, L. I. and O'Connor, M. B. (2009). The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science 326: 1403-1405. PubMed Citation: 19965758

Richards G., et al. (1999). The acquisition of competence to respond to ecdysone in Drosophila is transcript specific. Mech. Dev. 82(1-2): 131-139

Ruaud, A. F., Lam, G. and Thummel, C. S. (2010). The Drosophila nuclear receptors DHR3 and βFTZ-F1 control overlapping developmental responses in late embryos. Development 137(1): 123-31. PubMed Citation: 20023167

Sashihara, S., et al. (1996). Orphan nuclear receptor ROR alpha gene: isoform-specific spatiotemporal expression during postnatal development of brain. Brain Res Mol Brain Res 42 (1): 109-117

Sato, T. K., et al. (2004). A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43(4): 527-37. 15312651

Sluder, A. E., Lindbloom, T. and Ruvkun, G. (1997). The Caenorhabditis elegans orphan nuclear hormone receptor gene rhr-2 functions in early embryonic development. Dev. Biol. 184: 303-319

Steinhilber, D., et al. (1995). The nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes. J Biol Chem 270 (13): 7037-7040

Steinmayr, M., Andre, E., Conquet, F., Rondi-Reig, L., Delhaye-Bouchaud, N., Auclair, N., Daniel, H., Crepel, F., Mariani, J., Sotelo, C. and Becker-Andre, M. (1998). staggerer phenotype in retinoid-related orphan receptor alpha-deficient mice. Proc. Natl. Acad. Sci. 95: 3960-3965. 9520475

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

Sun, Z., et al. (2000). Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288(5475): 2369-2373. 10875923

Tini, M., Fraser, R. A., and Giguere, V. (1995). Functional interactions between retinoic acid receptor-related orphan nuclear receptor (ROR alpha) and the retinoic acid receptors in the regulation of the gamma F-crystallin promoter. J Biol Chem 270 (34): 20156-20161

Tsutsui, M., et al. (2006). Development of genetically engineered mice lacking all three nitric oxide synthases. J. Pharmacol. Sci. 102: 147-154. PubMed Citation: 17031076

Tsutsui, M., et al. (2009). Nitric oxide synthases and cardiovascular diseases: insights from genetically modified mice. Circ. J .73: 986-993. PubMed Citation: 19430166

Vorobyeva, N. E., et al. (2009). Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex. Proc. Natl. Acad. Sci. 106(27): 11049-54. PubMed Citation: 19541607

Vorobyeva, N. E., et al. (2011). SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation. Cell Cycle 10(11): 1821-7. PubMed Citation: 21519192

Vorobyeva, N. E., Nikolenko, J. V., Nabirochkina, E. N., Krasnov, A. N., Shidlovskii, Y. V. and Georgieva, S. G. (2012). SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing. Nucleic Acids Res 40: 7319-7331. PubMed ID: 22638575

White, K. P., et al. (1997). Coordination of Drosophila metamorphosis by two ecdysone-induced nuclear receptors. Science 276: 114-117. PubMed ID: 9082981

White, K., et al. (1999). Microarray analysis of Drosophila development during metamorphosis. Science 286: 2179-2184. PubMed ID: 10591654

Hormone receptor-like in 46: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 August 2023 

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.