InteractiveFly: GeneBrief

Ecdysone-induced protein 75B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Ecdysone-induced protein 75B

Synonyms - E75, E75A, E75B and E75C

Cytological map position - E75A8--B2

Function - Transcription factor

Keywords - molting

Symbol - Eip75B

FlyBase ID: FBgn0000568

Genetic map position -

Classification - hormone receptor

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Jaumouille, E., Machado Almeida, P., Stahli, P., Koch, R. and Nagoshi, E. (2015). Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr Biol 25: 1502-1508. PubMed ID: 26004759
Summary:
Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Rabinovich, D., Yaniv, S.P., Alyagor, I. and Schuldiner, O. (2016). Nitric oxide as a switching mechanism between axon degeneration and regrowth during developmental remodeling. Cell 164: 170-182. PubMed ID: 26771490
Summary:
During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), this study found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. It was found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.

Xiong, X.P., Kurthkoti, K., Chang, K.Y., Li, J.L., Ren, X., Ni, J.Q., Rana, T.M. and Zhou, R. (2016). miR-34 modulates innate immunity and ecdysone signaling in Drosophila. PLoS Pathog 12: e1006034. PubMed ID: 27893816
Summary:
microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. This study shows that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, there exists a mutual repression between miR-34 expression and ecdysone signaling, and miR-34 acts as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, the cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34 were identified. Taken together, these data enrich the repertoire of immune-modulating miRNAs in animals, and provide new insights into the interplay between steroid hormone signaling and innate immunity.
BIOLOGICAL OVERVIEW

Isoform-specific null mutations were used to define the functions of three orphan members of the nuclear receptor superfamily: E75A, E75B, and E75C. These proteins are encoded by the E75 early ecdysteroid-inducible gene (officially termed Ecdysone-induced protein 75B). Mutants for the isoform E75B are viable and fertile, while E75C mutants die as adults. In contrast, E75A mutants have a reduced ecdysteroid titer during larval development, resulting in developmental delays, developmental arrests, and molting defects. Remarkably, some E75A mutant second instar larvae display a heterochronic phenotype in which they induce genes specific to the third instar and pupariate without undergoing a molt. It is proposed that ecdysteroid-induced E75A expression defines a feed-forward pathway that amplifies or maintains the ecdysteroid titer during larval development, ensuring proper temporal progression through the life cycle (Bialecki, 2002).

Ecdysteroids function as key temporal signals in Drosophila, directing each postembryonic transition in the life cycle. Ecdysteroid pulses at 1 day intervals during the first and second larval instars trigger molting of the cuticle, accommodating the growth that occurs during these stages. A high-titer ecdysteroid pulse 2 days after the molt to the third instar triggers puparium formation, signaling the onset of prepupal development and metamorphosis. This is followed by another ecdysteroid pulse, approximately 10 hr after puparium formation, which triggers adult head eversion and the prepupal-pupal transition. A neuropeptide signal from the brain to the endocrine organ of the insect, the ring gland, triggers the release of relatively inactive ecdysteroids into the hemolymph that are converted by peripheral tissues into more active forms of the hormone, primarily 20-hydroxyecdysone, also known as 20E (Bialecki, 2002).

The ecdysteroid signal is transduced by a heterodimer of two members of the nuclear receptor superfamily, Ecdysone receptor and the RXR ortholog, Ultraspiracle. This hormone/receptor complex activates cascades of gene expression, as first defined by studies of the puffing patterns of the giant larval salivary gland polytene chromosomes. Ecdysteroids directly induce the formation of about a half dozen early puffs. The protein products of these puffs induce more than 100 late puffs scattered throughout the genome. The late puff products, in turn, are thought to act as effectors that direct appropriate biological responses to each ecdysteroid pulse during development (Bialecki, 2002).

Molecular characterization of three early puff genes has shown that they encode transcription factors, fulfilling a central prediction of the hierarchical model of ecdysteroid action. The Broad-Complex (BR-C), responsible for the 2B5 early puff, is a complex genetic locus that encodes a family of zinc finger proteins. Null mutations that inactivate all three essential BR-C subfunctions leads to prolonged third instar larvae that fail to pupariate, while mutations that affect only a single subfunction result in defects in imaginal disc morphogenesis, larval tissue cell death, and lethality during prepupal and pupal stages. BR-C mutations also have widespread effects on early and late ecdysteroid-inducible gene expression, consistent with a central role for this gene in transducing the ecdysteroid signal (Bialecki, 2002).

The two best-characterized early puffs, at 74EF and 75B, also encode ecdysteroid-inducible transcription factors. Ecdysone-induced protein 74EF (E74), from the 74EF puff, consists of two overlapping transcription units, E74A and E74B, that encode proteins containing an identical ETS DNA binding domain. E74 mutants display lethality during prepupal and pupal stages, with defects in adult head eversion and leg morphogenesis as well as defects in ecdysteroid-regulated gene expression (Bialecki, 2002).

The E75 ecdysteroid-inducible gene from the 75B early puff encodes three protein isoforms designated E75A, E75B, and E75C (Segraves, 1990). These proteins contain both a canonical DNA binding domain and a ligand binding domain that define members of the nuclear receptor superfamily, although these proteins are referred to as orphan nuclear receptors because a corresponding hormonal ligand has not yet been identified. Each E75 isoform is characterized by a unique N-terminal sequence encoded by a distinct 5' exon (Segraves, 1990). These 5' exons splice to a common set of five 3' exons for E75A and E75C, while E75B shares only the last four 3' exons. As a result of this arrangement, E75B contains only one of the two E75 zinc fingers and is thus incapable of binding DNA. E75B can, however, heterodimerize with the DHR3 ecdysteroid-inducible orphan nuclear receptor and has been detected on salivary gland polytene chromosomes by antibody stains (White, 1997), indicating that it may function at the level of target gene regulation (Bialecki, 2002).

Relatively little is known about E75 functions during the Drosophila life cycle. Evidence from biochemical and ectopic expression studies indicates that E75B can act as a repressor of the ßFTZ-F1 competence factor during metamorphosis (White, 1997). Germline clones of E75 null mutants, missing all three isoforms, led to arrest during mid-oogenesis, similar to the phenotype of EcR mutant germline clones. A zygotic loss of E75 function results in midgut morphogenesis defects during embryogenesis (Bilder, 1995). Genetic studies of individual E75 isoforms, however, and more detailed phenotypic studies at other stages of the life cycle, have not been reported. This study examined the phenotypes of null mutations specific to E75A, E75B, and E75C, as well as a mutant in which none of the three E75 isoforms was expressed (Bialecki, 2002).

E75B mutants are shown to be viable and fertile, suggesting that this gene functions in a redundant pathway during development, while E75C mutants die as adults. In contrast, most E75A mutants die as delayed second instar larvae with a reduced ecdysteroid titer, or arrest during the molt to the third instar. Remarkably, some E75A mutant second instar larvae express genes characteristic of the third instar and pupariate without progressing through a molt, indicating that molting can be uncoupled from the onset of metamorphosis. This study provides a new direction for understanding the functions of early ecdysteroid-inducible regulatory genes, positioning the E75A orphan nuclear receptor upstream from the signal that induces its expression, defining its action in a feed-forward pathway to amplify or maintain ecdysteroid titers during Drosophila larval development (Bialecki, 2002).

E75B null mutants show no detectable phenotypes. In addition, the expression of 16 ecdysteroid-regulated genes at the onset of metamorphosis is normal in these mutants. Interestingly, ßFTZ-F1 is among the genes that remain unaffected by the E75B mutation. Ectopic expression and biochemical studies have shown that E75B can directly interact with the DHR3 orphan nuclear receptor and thereby block its ability to induce ßFTZ-F1 in mid-prepupae (White, 1997). These observations led to the proposal that the timing of ßFTZ-F1 expression is dependent on appropriate decay of the E75B repressor. No reproducible temporal shift, however, was found in ßFTZ-F1 expression in E75B mutant prepupae, indicating that, while E75B may be sufficient to repress ßFTZ-F1, it is not necessary for this response. This conclusion, combined with the absence of obvious phenotypic effects caused by the E75B mutation, raises the possibility that E75B acts in a functionally redundant pathway. E78B is an ideal candidate for exerting this redundant activity. Like E75B, E78B encodes a homolog of the vertebrate Rev-Erb orphan nuclear receptor (NR1D1), and is a truncated isoform that lacks a DNA binding domain (Stone, 1993). E78B is expressed in synchrony with E75B in early prepupae (Karim, 1992; Stone, 1993). In addition, E78B null mutants display no detectable phenotypes, like E75B mutants, suggesting that its function is complemented by another factor. An effort is currently underway to inactivate both E75B and E78B in order to determine whether they act in a redundant manner during development (Bialecki, 2002).

E75C mutants die as pharate adults or within a few days following eclosion. These animals are morphologically normal except for black spots that cover up to one quarter of the eye, but are weak, unable to fly, and severely uncoordinated. Earlier development in these mutants appears to proceed normally. Consistent with this observation, most of the 16 ecdysteroid-regulated transcripts examined in E75C mutant late third instar larvae and prepupae displayed normal temporal patterns of expression. The expression of four transcripts, however, fails to be maintained through the prepupal-pupal transition in E75C mutants: E75B, Fbp-1, L71-1, and L71-3. This coordinate misregulation suggests that the brief peak of ecdysteroid-induced E75C expression in ~10 hr prepupae is required for the continued expression of a subset of ecdysteroid target genes. It is unclear, however, whether these relatively subtle effects on gene expression might be causally related to the late developmental defects observed in E75C mutants (Bialecki, 2002).

Similarities between the E75C adult phenotype and the phenotype exhibited by hypomorphic dare alleles raises the possibility that the adult lethality of E75C mutants may result from an ecdysteroid deficiency. dare plays a pivotal role in Drosophila ecdysteroid biosynthesis. Dare is an ortholog of adrenodoxin reductase, a mammalian enzyme that plays a central role in vertebrate steroid hormone biosynthesis by transferring electrons to all known mitochondrial cytochrome P450s. dare mutant adults are unable to walk or fly, exhibiting twitching of the legs and wings, and die within a week following eclosion (Freeman, 1999). These dare mutant phenotypes appear to arise from progressive degeneration of the CNS, starting at adult eclosion. Future studies could provide a basis for determining whether E75C and dare might function together to control ecdysteroid titers during pupal and adult development (Bialecki, 2002).

Most E75A mutants display defects during the second larval instar, either failing to molt to the third instar, arresting at the molt, or forming prepupae directly from the second instar. E75A mutant larvae develop asynchronously and can molt up to 1 day late. Larvae that do not molt can live for up to a week before dying. These phenotypes resemble those seen with a temperature-sensitive dre-4 mutant as well as hypomorphic alleles of itpr and null alleles of dare. The dre4 gene has not yet been isolated, although its function is required for ecdysteroid pulses throughout the life cycle (Sliter, 1992). itpr encodes an intracellular calcium channel, the inositol 1,4,5-triphosphate (IP3) receptor, that is expressed in the ring gland and appears to be required for ecdysteroid biosynthesis. Consistent with their proposed functions, the molting defects in itpr and dare mutant larvae can be rescued by providing ecdysteroids in the culture medium. Similarly, E75A mutant second instar larvae can molt to the third instar when fed ecdysteroids, suggesting that an ecdysteroid deficiency is the primary cause of the observed developmental defects at this stage. More direct evidence for this conclusion is provided by an enzyme immunoassay which indicates that the ecdysteroid pulse is essentially eliminated in E75A mutant second instar larvae. The ring gland, as well as other larval tissues, appears normal in E75A mutants, indicating that, like other early genes, E75A does not play a role in their growth or development. Rather, it is concluded that E75A is required for appropriate ecdysteroid biosynthesis or release during larval development (Bialecki, 2002).

While these studies define an essential role for E75A in directing ecdysteroid pulses during larval development, they do not preclude additional functions for this gene during the life cycle -- functions that could be masked by the early lethality of E75A mutants. Like other early genes, E75A is expressed widely in third instar larvae, in contrast to itpr and dare, which are expressed primarily in the ring gland. E75A transcripts have been detected in the salivary glands, gut, Malpighian tubules, fat bodies, and imaginal discs (Huet, 1993; Segraves, 1988), reflecting the widespread expression of E75A protein detected by antibody stains, including abundant expression in the ring gland. E75A protein is also bound to multiple sites in the larval salivary gland polytene chromosomes (Hill, 1993). These observations suggest that E75A may play additional roles beyond those revealed by this study, possibly in a redundant manner with other E75 isoforms or in combination with other early ecdysteroid-inducible genes (Bialecki, 2002).

The E75 mutant that is missing all three E75 isoforms dies after a prolonged first instar, failing to molt to the second instar, similar to dre4 and itpr null mutants. In addition, the earliest phenotypes observed in E75 common region mutants -- embryonic lethality with head involution and midgut morphogenesis defects -- resemble the lethal phenotypes of disembodied (dib) mutant embryos, mutants that are missing a key cytochrome P450 required for ecdysteroid biosynthesis (Chavez, 2000). These observations indicate that the E75 locus may play an essential role in maintaining ecdysteroid titers through embryonic and larval development. The individual E75 isoforms, however, appear to contribute to this regulatory function in a redundant manner because the highly penetrant early lethality associated with the E75 common region mutation is not seen with mutations in any of the individual E75 isoforms. It is concluded that E75A and E75C are good candidates for exerting this redundant activity by virtue of their identical DNA binding domain (Bialecki, 2002).

Redundant interactions between E75A and E75C may explain the apparent stage specificity of the E75A mutant phenotypes. E75A and E75C mutants display no defects at puparium formation or head eversion, key developmental transitions triggered by ecdysteroid pulses at the onset of metamorphosis. Indeed, most E75A mutants progress through to pupal stages once they have passed beyond the second instar. Similarly, exposure of E75A mutant second instar larvae to a single 6 hr treatment with ecdysteroids is sufficient to rescue many animals through to pupal and pharate adult stages. In addition, E75A mutant L2 prepupae appear to execute relatively normal changes in ecdysteroid titer at the onset of metamorphosis, as indicated by the patterns of E74 and ßFTZ-F1 transcription. It is thus proposed that E75A and E75C act in a redundant manner to maintain ecdysteroid titers during the onset of metamorphosis, explaining why mutations in either function survive these stages in the life cycle (Bialecki, 2002).

E75A mutant second instar larvae that fail to molt continue to grow, approaching the size of a wild-type late third instar larva. Remarkably, these delayed second instar larvae express markers that are specific to the latter half of the third instar -- the fat body-specific Fbp-1 larval serum protein receptor gene and the Sgs-4 salivary gland glue protein gene. These changes in gene expression, in apparent preparation for metamorphosis, are consistent with the ability of these second instar larvae to pupariate and progress through head eversion, dying as early pupae. E75A mutant L2 prepupae display normal temporal patterns of E74 and ßFTZ-F1 transcription through prepupal and early pupal stages, with an ~2 hr delay relative to control animals. These observations suggest that these mutants can reset their endocrinological clock to execute the proper ecdysteroid pulses that trigger puparium formation and pupation (Bialecki, 2002).

The ability of E75A mutant animals to pupariate directly from the second instar indicates that molting can be uncoupled from progression through the onset of metamorphosis. This appears to be a manifestation of the reduced ecdysteroid titer in these mutants, since pupariating second instar larvae have also been reported in dre4 and itpr mutants, although they were not characterized in any detail. The reduced ecdysteroid titer in E75A mutant second instar larvae apparently causes these animals to miss this cue that would normally trigger the molt to the third instar. In spite of this, however, these animals can still acquire third instar identity and progress relatively normally, albeit with a significant developmental delay that is evident both in terms of E74A induction in staged larvae, as well as the time to puparium formation, which is at least 16 hr later than puparium formation in wild-type animals. This conclusion suggests that larval molting is an epidermal response that has few, if any, consequences for temporal progression throughout the rest of the organism. Moreover, the observation that a reduced ecdysteroid titer can lead to a heterochronic phenotype at the onset of metamorphosis defines a critical role for ecdysteroid pulses in defining not just timing but also the character of the major developmental transitions in the fly life cycle (Bialecki, 2002).

The observation that E75A expression is induced directly by ecdysteroids (Segraves, 1990), combined with its requirement for appropriate ecdysteroid titers during larval development, leads to the proposal that E75A functions in a feed-forward pathway to maintain or amplify ecdysteroid pulses during Drosophila larval development. The most direct means by which E75A could exert this regulatory function would be through transcriptional control of genes that encode steroidogenic enzymes. The transcription of dare and dib in E75A mutant second instar larvae was examined as a means of testing this hypothesis, but no effects on their expression were detected. Although dare and dib are the only members of the ecdysteroid biosynthetic pathway that have been characterized at the molecular level, there are other genes in this pathway that could be dependent on E75A function. These include members of the so-called 'Halloween' class of genes, such as spook, shroud, and phantom (Chavez, 2000), which display lethal mutant phenotypes similar to those of dib (Bialecki, 2002).

The reduced level of ßFTZ-F1 expression in E75A mutant second instar larvae provides a functional link to explain the molting defects in these animals. ßFTZ-F1 has been shown to be required for larval molting. Moreover, ßFTZ-F1 can directly regulate the Edg84A pupal cuticle gene, and ectopic overexpression of ßFTZ-F1 leads to an abnormal larval cuticle structure. Taken together, these observations suggest that ßFTZ-F1 plays a key role in controlling larval molts, directly regulating larval cuticle gene expression. The severe reduction in ßFTZ-F1 expression in E75A mutant larvae is thus consistent with the inability of these animals to molt. It is also concluded that E75A does not directly regulate ßFTZ-F1, since molting can be rescued by feeding ecdysteroids to E75A mutant larvae. This experiment places ßFTZ-F1 downstream from ecdysteroid signaling and E75A expression, indirectly dependent on E75A activity (Bialecki, 2002).

These studies of E75 provide a new direction for understanding early gene function within the ecdysteroid-triggered regulatory cascades. Previous work has indicated that early ecdysteroid-inducible genes operate downstream from the ecdysteroid receptor, coordinating the expression of secondary response late genes that, in turn, execute the appropriate biological responses to each hormone pulse during development. This characterization of E75 functions indicates that early genes not only transduce the ecdysteroid signal but can also affect the signal itself, through feedback regulation. Further studies of E75 should provide a molecular framework for understanding the genetic control of steroidogenesis in insects. In addition, the heterochronic phenotypes in E75A mutants provide a basis for understanding how key developmental landmarks, such as molting, are linked to temporal progression through the insect life cycle (Bialecki, 2002).

Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75

Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Previous work showed that ecdysone-induced protein 75 (E75, also known as Eip75B; NR1D3) contains heme constitutively bound to its ligand-binding domain (LBD), and that amino acids coordinately bound to the heme iron can be displaced in vitro by changes in redox state or the presence of nitric oxide (NO) gas (Reinking, 2005; Marvin, 2009). In turn, these structural changes negate the ability of E75 to repress transcription and to reverse the positive transcriptional activity of its heterodimer partner, Drosophila hormone receptor 3 (DHR3; also known as DHR46, NR1F4). This study examined whether these interactions are relevant in vivo and, if so, what their roles are. Using genetic and chemical manipulations, it was shown that nitric oxide is produced in the Drosophila prothoracic gland (PG), where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates (Cáceres, 2011).

Although previously documented effects of NO on cells and tissues are numerous, and many of the cytoplasmic mechanisms of action are well documented, this is the first demonstration of a direct effect via a transcription factor in vivo. In the PG, the findings indicate that E75 is the major nuclear mediator of NO function, as evidenced by the similarity and epistatic nature of E75, DHR3, and NOS phenotypes. A review of the literature also shows that these genes tend to be coexpressed in many other Drosophila tissues, where limited analyses also suggest shared functions. Thus, the interactions described in this study are likely to be relevant to numerous other tissues and processes (Cáceres, 2011).

This overlap in expression and functions is also true for the vertebrate NOS and Rev-erb/ROR orthologs. Examples of overlapping functions in vertebrates include their similar roles in lipid metabolism, gluconeogenesis, muscle differentiation, inflammation, circadian rhythm, PGC1α regulation, hypertension, and atherosclerosis. Strikingly, Nos triple-knockout mice that survive gestation are morbidly obese, exhibiting all aspects of metabolic syndrome (e.g., diabetes, hypertension, atherosclerosis), resulting in a maximum life span of 10 mo (Tsutsui, 2006; Tsutsui, 2009). Conversely, NO up-regulation via arginine supplementation yields a reciprocal phenotype, which includes increased lipolysis, fatty acid oxidation, mitochondrial biogenesis, glucose metabolism, and life span. These effects are similar to those seen upon genetic manipulation of the Rev-erbs and RORs (Duez, 2009a; Duez, 2009; Cáceres, 2011 and references therein).

In the case of E75, NO appears to act in two ways. NO blocks the ability of E75 to interfere directly with DHR3-mediated transcriptional activation. However, it also appears to block the ability of E75 to repress target genes independently of DHR3. In vertebrates, no direct interaction has yet been observed between Rev-erbs and RORs. NO does, however, block the ability of the Rev-erbs to repress target genes by preventing the recruitment of coactivators (Pardee, 2009). It is expected that this aspect of NO action is conserved in flies (Cáceres, 2011).

The achievement of critical weight at the end of the third larval instar coincides with a reduction in circulating levels of insulin-like peptides (ILPs) and consequential production of prothoracicotropic hormone (PTTH) peptide by neurons located within the larval brain. The axons of these PTTH-producing neurons extend to the surface of the PG, where binding of the secreted PTTH peptide to Torso receptor results in intracellular signaling (Rewitz, 2009). Intracellular outcomes include activation of Ras/Raf, MEK/Erk, and PKA, and, notably, the production of Calmodulin, NADH, and cytoplasmic Ca2+ influx. The latter three molecules are required cofactors for dNOS enzymatic activity. Hence, it is proposed that PTTH acts, in large part, through NOS activation (Cáceres, 2011).

The remarkable endoreduplication and growth of the PG due to loss of NOS expression is consistent with previous studies showing that NO has negative effects on cell growth. This activity has important implications and potential use in the control of oncogenic growth (Cáceres, 2011).

Interestingly, these effects of NO on PG size and the timing of metamorphosis contradict previous studies that had suggested that PG size is a key determinant of metamorphosis timing. In these studies, metamorphosis appeared to occur when full PG size was achieved. Here, PG size was up to six times larger than normal, with no metamorphosis. Similarly, Colombani (2005) found that altering PG size by manipulation of DMyc or Cyclin D expression could also increase PG size without triggering premature ecdysone production. Furthermore, metamorphosis can be induced prematurely by down-regulation of DHR4 without affecting PG size. These seemingly contradictory results are likely due to cross-talk within the insulin and ecdysone signaling pathways (Cáceres, 2011).

The bright-red color of these enlarged PGs is consistent with previous studies suggesting that E75 and the Rev-erbs also function as heme sensors (Cáceres, 2011).

NOS manipulation in the PG had major effects on lipid uptake and storage, leading to a nearly 20-fold increase in larval lipid content in the case of knockdown, or a nearly fivefold decrease when expressed prematurely. As disruption of EcR activity within fat body cells can also result in lipid overaccumulation (Colombani, 2005), the nonautonomous effects of PG NOS manipulation appear to be ecdysone-mediated. However, it is quite possible that the nearly 100-fold overall changes observed in lipid content may also be due to additional effects on feeding behavior, nutrient uptake, and other aspects of lipid metabolism (Cáceres, 2011).

The extended eating and fat deposition phenotype, caused by failure to produce ecdysone, may equate on many levels to processes underlying obesity and diabetes. Indeed, much as seen with Nos up-regulation, ecdysone supplementation in vertebrates results in decreased appetite, cholesterol synthesis, and weight gain, while at the same time increasing muscle mass and endurance. Given these apparent abilities to switch fat cell activity from lipid storage to lipid mobilization, a better understanding of the signals and mechanisms underlying NO and ecdysone-mediated effects should provide new insights and possible uses in metabolism based disorders (Cáceres, 2011).

Given the previously demonstrated roles for NO, Rev-erbs, and RORs in the control of circadian clocks, and those shown in this study for developmental timing, it is quite possible that NO, Rev-erbs, and RORs also serve general roles in the matching of diurnal, lunar, and seasonal inputs (i.e., light, temperature, and food availability) with appropriate feeding behaviors, metabolism, and developmental progression. Disruption of these functions leads to metabolic, sleep, stress, immune, and hypertension disorders. Hence, further elucidation of ROR, Rev-erb, and NOS interactions in neuroendocrine tissues should provide new insights into how these temporal and metabolic processes are linked and coordinated, and new ways to prevent or treat their related disorders (Cáceres, 2011).


REGULATION

Transcriptional Regulation

The Hr46/DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. Hr46 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Hr46 directly contributes to both of these regulatory responses. Hr46 protein binds to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory proteins, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Hr46 represses E74A, and to a lesser extent E74B, and it also represses BR-C, E75A, and E78B. Hr46 activates betaFTZ-F1. Three Hr46 binding sites are present downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of Hr46 regulation. Ectopic expression of Hr46 reveals that the polytene chromosome binding pattern is of functional significance. Hr46 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. Hr46 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae. This study also provides evidence for direct cross-regulation among orphan members of the nuclear receptor superfamily and further implicates these genes as critical transducers of the hormonal signal during the onset of Drosophila metamorphosis (Lam, 1997).

The beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses, adult head eversion, leg elongation and salivary gland death, in response to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. Both E74A and E75A, whose levels of expression are normally increased several orders of magnitude by ecdysone, are significantly affected in betaFTZ-F1 mutants. The severity of these effects correlates with the intensity of polytene chromosome staining by FTZ-F1 antibodies. The Br-C locus is only weakly stained, while E74 is strongly stained, and E75 is the most intensely stained site in the genome. It thus appears that betaFTZ-F1 exerts specificity to the degree to which it can enhance the ecdysone-induction of different promoters. The E93 early gene is also submaximally induced in betaFTZ-F1 mutants, consistent with the proposal that this stage-specific response is dependent on betaFTZ-F1 function. In contrast, the levels of Ecdysone receptor and Ultraspiracle mRNA are not significanty affected by betaFTZ-F1. EDG84A, a gene that encodes a pupal cuticle protein that is specifically expressed in the imaginal discs of mid-prepupae, contains a betaFTZ-F1 binding site upstream from the start site, and EDG84A transcription is delayed and reduced in betaFTZ-F1 mutants. Thus this study defines beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence (Broadus, 1999).

Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).

The nuclear localization of E93 in larval salivary glands provided an opportunity to determine if E93 binds to the salivary gland polytene chromosomes and, if so, to identify the sites bound by the protein. Salivary glands were dissected 12-14 hr after puparium formation, fixed, squashed, and photographed to acquire accurate cytology of the banding and puffing patterns for mapping. The chromosomes were then stained with affinity-purified E93 antibodies, and these patterns were compared with the original set of photographs to allow accurate mapping of the bound sites. E93 clearly binds to the polytene chromosomes in a reproducible and site-specific manner and is consistently detected at 65 chromosome sites, many of which contain ecdysone-regulated genes or programmed cell death genes. Among these sites are the 74EF and 75B early puffs, which contain the E74 and E75 ecdysone-inducible genes, as well as the 93F puff, which contains E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and contain the programmed cell death genes dredd, crq, dcp-1, and drICE, respectively. The 2B5 early puff, containing the BR-C ecdysone-inducible gene, and 75CD, containing βFTZ-F1 and the programmed cell death genes rpr, hid, and grim, were not bound by E93. These data indicate that E93 may directly regulate the genes in bound chromosome loci and may either encode a site-specific DNA binding protein or a chromatin-associated protein that functions as a transcriptional regulator (Lee, 2000).

The observations that E93 is essential for salivary gland cell death and that E93 protein binds to specific sites in the salivary gland polytene chromosomes suggest that E93 may regulate the transcription of target genes that function in steroid-triggered programmed cell death. If this hypothesis is true, then E93 mutations should impact the transcription of genes that reside in salivary gland chromosome loci bound by E93. Salivary glands were dissected from staged late third instar larvae, prepupae, and pupae of control and mutant animals. Total RNA extracted from these tissues was analyzed by Northern blot hybridization. E93 mutations have little or no effect on the timing and levels of BR-C, E74, and E75A transcription in the salivary glands of late third instar larvae and early prepupae. However, the level of expression of each of these regulatory genes is significantly reduced or absent in salivary glands 10-24 hr following puparium formation. Although the smaller E74B transcript is induced, the larger E74A RNA is not detected following the prepupal pulse of ecdysone. The levels of EcR expression in late third instar larval and prepupal salivary glands are not altered by E93 mutations, although its timing is delayed by 4-6 hr at the prepupal to pupal transition. Like EcR, βFTZ-F1 transcription is delayed but the level of this mRNA is not altered in E93 mutant salivary glands. A similar delay is observed in the parental flies that were used for mutagenesis, indicating that this effect is due to the genetic background. The induction of EcR and E74B in E93 mutant prepupae, as well as the successful completion of adult head eversion, indicates that the prepupal pulse of ecdysone occurs in these mutant animals, signaling the prepupal-pupal transition (Lee, 2000).

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).

Ecdysone receptor (EcR) suppresses lipid accumulation in the Drosophila fat body via transcription control

Lipid metabolism drastically changes in response to the environmental factors in metazoans. Lipid is accumulated at the food rich condition, while mobilized in adipocyte tissue in starvation. Such lipid mobilization is also evident during the pupation of the insects. Pupation is induced by metamorphosis hormone, ecdysone via ecdysone receptor (EcR) with lipid mobilization, however, the molecular link of the EcR-mediated signal to the lipid mobilization remains elusive. To address this issue, EcR was genetically knocked-down selectively in 3rd instar larva fat body of Drosophila, corresponding to the adipocyte tissues in mammalians, that contains adipocyte-like cells. In this mutant, lipid accumulation was increased in the fat body. Lipid accumulation was also increased when knocked-down of taiman, which served as the EcR co-activator. Two lipid metabolism regulatory factor, E75B and adipose (adp) as well as cell growth factor, dMyc, were found as EcR target genes in the adipocyte-like cells, and consistently knock-down of these EcR target genes brought phenotypes in lipid accumulation supporting EcR function. These findings suggest that EcR-mediated ecdysone signal is significant in lipid metabolism in insects (Kamoshida, 2012).

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

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

An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila

Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. This study reports a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which was identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, Clock (Clk), and may also affect circadian output. Per inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of Per on Clk transcription. The Ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila (Kumar, 2014).

Transcriptional Regulation via Nuclear Receptor Crosstalk Required for the Drosophila Circadian Clock

Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

This study has identified the nuclear receptors E75 and UNF as components of the molecular clocks in the s-LNvs. E75 is the closest homolog of mammalian REV-ERB α and REV-ERB β, which play important roles in the molecular clock feedback loops. In contrast with Rev-Erb α/β, which represses transcription, the results demonstrated that E75 is neither a potent repressor nor a strong activator but potentiates the activation of per transcription by UNF. Despite these mechanistic divergences, the notion that Rev-Erb homologs are integral to the molecular oscillators in both Drosophila and mammals highlights the significance of transcriptional regulations via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Rev-Erb α and Rev-Erb α are rhythmically transcribed by the CLOCK/BMAL1 transcriptional activators, and REV-ERBs periodically repress the transcription of Bmal1, thereby forming a feedback loop to ensure robust molecular oscillations of the mammalian clock. A previous study demonstrated that E75 is a cycling target of Clk/Cyc in the fly head (Kumar, 2014). Because E75 has three isoforms, it was not possible to determine whether any of the isoforms were rhythmically expressed in the LNvs from the RNA profiles of the isolated LNvs. Nonetheless, the results indicate that E75 together with Unf (which is not a Clk/Cyc target) reinforces the main loop of the core fly clock composed of Clk/Cyc and Per/Tim through a feedforward mechanism, showcasing the mechanistic parallels between fly and mammalian clocks (Jaumouille, 2015).

E75 has been demonstrated to covalently bind to heme, and its binding appears to stabilize the E75 and facilitates the binding of nitric oxide (NO) and carbon monoxide (CO). The NO/CO binding to E75 modulates the transcriptional activity of its known heterodimeric partner DHR3. To test whether similar mechanisms are involved in the action of E75 in the s-LNvs, attempts were made to disrupt cellular heme metabolism by knocking down the enzymes in the heme biosynthesis pathway, coproporphyrinogen oxidase (Coprox) and protoporphyrinogen oxidase (Ppox), and the key enzyme in the heme degradation pathway, heme oxygenase (Ho). These experiments were inconclusive, as no effect on the behavioral rhythms were observed by any knockdown with Pdf-GAL4, and knockdown with Tim-GAL4 was lethal (Jaumouille, 2015).

S2 cell experiments showed that Unf is a transcriptional activator of per, and concurrent expression of E75 and Unf increases the turnover of Unf binding to per regulatory sequences. This high turnover is correlated with higher transcriptional activity. The finding that E75 acts through Unf on transcription is consistent with in vivo data: (1) depletion of both Unf and E75 in adult LNvs abolishes the behavioral rhythms; (2) E75 overexpression has no effect on the behavioral rhythms; and (3) E75 overexpression does not rescue Unf knockdown. Although unf mRNA levels do not oscillate, Unf protein levels cycle in the s-LNvs, peaking at zeitgeber time (ZT)2 and lowest at ZT14. Low Unf levels may reflect the degradation as a consequence of higher transcriptional activity. Indeed, per is most actively transcribed around ZT13 when Unf levels are minimum in the s-LNvs. Nonetheless, downregulation and arrhythmia of Per levels in the s-LNvs is most probably not the sole cause of the altered locomotor rhythms in the Unf knockdown, E75 knockdown, and Unf/E75 double knockdown. A recent study showed the implication of E75 in the repression of Clk transcription, although the current results are not in concordance with this observation probably due to the differences in the reagents used for E75 knockdown and the timing of knockdown. Deciphering whether E75 and Unf heterodimerize or bind to adjacent sequences, how they cooperate with Clk/Cyc, and whether any ligand is involved in their transcriptional regulation will yield new insights into the diverse mode of nuclear receptor crosstalk and their critical roles in circadian biology (Jaumouille, 2015).

Protein Interactions

The E75B receptor, which lacks a complete DNA binding domain, inhibits the inductive function of Hr46/DHR3 on ßFTZF1 by forming a complex with DHR3 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. Thus the repressive function of Hr46 does not involve binding to DNA but instead involves physical interaction with the Ecdysone receptor. E75B fails to bind DNA in the absence of HR46. Thus E75B acts like a co-repressor with HR46, rather than as a competitor with Hr46 for DNA binding; the restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).

rigor mortis encodes a nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. A novel nuclear receptor interacting protein is described, encoded by rigor mortis (rig), that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation -- phenotypes that resemble those seen in EcR, usp, E75A and ßFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and ßFTZ-F1. The ligand binding domain of ßFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development (Gates, 2003).

Mutations in rig result in prolonged second and third instar larval stages, defects in molting, larval lethality and duplicated mouth parts. These phenotypes are characteristic of defects in ecdysone signaling, suggesting a critical role for rig in ecdysone responses during larval development. Two classes of genes produce mutant phenotypes that resemble those seen in rig mutant animals: those required for ecdysone biosynthesis or release -- including ecdysoneless (ecd), dare and itpr -- and those encoding nuclear receptors that mediate the ecdysone signal -- EcR, usp, E75A, and ßFTZ-F1. Unlike ecdysone-deficient mutants, the lethal phenotypes of rig mutants cannot be rescued by feeding 20E, indicating that ecdysone is not limiting in these animals and that rig acts downstream from hormone biosynthesis or release. Rather, it is proposed that Rig is functioning as a nuclear receptor cofactor, based on five lines of evidence. (1) The lethal phenotypes of rig mutants are very similar to those defined for EcR, usp, E75A and ßFTZ-F1, although all of these nuclear receptor genes are expressed in an essentially normal manner in rig mutant larvae. (2) rig mutants display a defect in the ecdysone-triggered switch in E74 isoform expression that is characteristic of reduced ecdysone signaling, indicating that rig is required for the appropriate expression of specific ecdysone-inducible genes. (3) These effects on gene expression are likely to be indirect as the predicted Rig protein sequence contains multiple protein-protein interaction domains and no known DNA-binding motifs. (4) Rig protein can interact physically with several Drosophila nuclear receptors, including EcR, USP and ßFTZ-F1, all of which have mutant phenotypes in common with rig mutants. (5) Rig protein shuttles between the cytoplasm and nucleus of larval cells in a manner similar to the active subcellular redistribution that has been reported for known Drosophila and vertebrate nuclear receptor cofactors (Gates, 2003).

Five Drosophila nuclear receptor cofactors have been identified to date: Alien, SMRTER, MBF1, Taiman and Bonus. Of these, only bonus appears to have activities in common with rig, although relatively limited genetic studies have been undertaken for most of these cofactors. No mutants have been characterized for SMRTER or Alien, which act as co-repressors in tissue culture transfection assays. MBF1 null mutants are viable and display a strong genetic interaction with tdf/apontic mutants: this indicates a role in tracheal and nervous system development. Somatic clones of taiman mutants reveal a role in border cell migration during oogenesis. In contrast, bonus mutants display first instar larval lethality as well as defects in salivary gland cell death and cuticle and bristle development, implicating a role for bonus in ecdysone responses during development. Also like rig, bonus mutations result in gene-specific defects in ecdysone-regulated transcription, and Bonus protein can interact with a range of Drosophila nuclear receptors, including EcR, USP, SVP, DHR3 and FTZ-F1. Bonus, however, interacts with these receptors in an AF-2-dependent manner, unlike Rig. Moreover, the larval lethal phenotypes of rig mutants do not resemble those reported for bonus mutants and, unlike Rig, Bonus protein appears to be exclusively nuclear in both larval and imaginal tissues. Further work is required to determine whether bonus and rig might act together to regulate ecdysone response pathways (Gates, 2003).

Rig is distinct from all known Drosophila nuclear receptor cofactors in that it is not part of an evolutionarily conserved protein family. Alien, SMRTER, MBF1, Taiman and Bonus all have vertebrate homologs, and Taiman and Bonus are the fly orthologs of the well characterized vertebrate nuclear receptor cofactors AIB1 and TIF1, respectively. In contrast, Rig does not contain identifiable enzymatic activities nor the conserved functional domains that define most nuclear receptor cofactors. BLAST searches with the Rig protein sequence did not reveal any closely related sequences in other organisms, although the top hits, which show limited homology in the WD-40 repeats, are in factors known to modify chromatin, including human histone acetyltransferase type B subunit 2 (RBBP-7) and chromatin assembly factor 1 (CAF-1) (Gates, 2003).

The WD-40 repeats that comprise about half of the Rig protein sequence are likely to play an important role in its activity. Consistent with this proposal, an N-terminal fragment of Rig, containing two WD-40 repeats but missing the LXXLL motif (amino acids 1-300), is capable of interacting with GST-DHR3 and GST-USP, suggesting that these repeats are sufficient for Rig-nuclear receptor interactions. WD-40 repeats provide multiple surfaces for protein-protein interactions and have been identified in over 150 proteins that function in a wide range of processes, including cytoskeleton assembly, transcriptional regulation, and pre-mRNA processing. In Drosophila, WD-40 repeats are associated with several transcriptional regulators, including the p85 subunit of TFIID, the Polycomb group protein encoded by extra sex combs, and the Groucho corepressor. In addition, a WD-40 repeat protein, TBL1, has been identified as part of a multiprotein complex with thyroid hormone receptor that contains the SMRT nuclear receptor corepressor and HDAC-3. The presence of these sequences in Rig may thus provide a scaffold for protein-protein interactions that could mediate the formation of multiprotein transcriptional complexes on ecdysone-regulated promoters. Further biochemical studies of Rig should provide insights into the significance of its WD-40 repeats as well as a foundation for understanding how Rig exerts its effects on transcription (Gates, 2003).

It is not clear how Rig expression in the brain, imaginal discs and salivary glands of second and third instar larvae is related to the lethal phenotypes of rig mutants, although neuroendocrine signaling is clearly required for molting, a process that is defective in rig mutant larvae. The subcellular localization of Rig protein at later stages, however, correlates with the distinct fates of larval and imaginal cells during metamorphosis. Rig protein appears to be restricted to the cytoplasm of cells that are fated to form parts of the adult fly, including neuroblasts, imaginal discs, and the imaginal islands of the larval midgut. In contrast, Rig shows dynamic changes in its subcellular distribution in larval salivary gland and midgut cells, both of which undergo steroid-triggered programmed cell death during metamorphosis. It is possible that these differences in subcellular localization could contribute to the distinct fates of these tissues in response to ecdysone signaling (Gates, 2003).

In addition to this spatial correlation, there is also a temporal correlation between the times at which Rig protein shuttles between the cytoplasm and nucleus in larval tissues and the coordinated changes in ecdysone-regulated gene expression that occur during the third instar. The switch from cytoplasmic to nuclear localization in larval salivary glands and midguts occurs at approximately the same time, 24-30 hours after the second-to-third instar larval molt, suggesting that Rig may be responding to a common temporal signal. Cell type-specific factors, however, must also contribute to this regulation as Rig is localized to the nucleus of only a subset of cells in the larval midgut. Interestingly, this protein redistribution correlates with a poorly understood event that is represented by widespread changes in ecdysone-regulated gene expression, called the 'mid-third instar transition.' It is possible that the cytoplasmic-to-nuclear transport of Rig in larval tissues contributes to the regulation of this response, which prepares the animal for metamorphosis one day later. Similarly, Rig returns to the cytoplasm of salivary gland cells at puparium formation, in synchrony with the widespread changes in ecdysone-regulated gene expression associated with the onset of metamorphosis. This translocation, however, is not seen in the larval midgut, where Rig protein remains in the nucleus of some cells. Rig shuttling thus appears to be differentially controlled in both a temporally and spatially restricted manner, correlating with major switches in ecdysone-regulated transcription. The observation that the first of these shifts in subcellular distribution occurs during the major lethal phase of rig mutants -- the mid-third instar -- suggests that these intracellular movements contribute to the critical functions of Rig during development (Gates, 2003).

Interestingly, several recent reports have described the subcellular redistribution of nuclear receptor cofactors in both vertebrate and Drosophila cells. The p/CIP vertebrate nuclear receptor coactivator is differentially distributed within the cells of the mouse female reproductive organs. For example, p/CIP is detected primarily in the nuclei of highly proliferative follicular cells while it is most abundant in the cytoplasm of terminally differentiated cells of the corpus luteum. p/CIP displays active nucleocytoplasmic shuttling in response to growth factors in cell culture, and interacts directly with the microtubule network in the cytoplasm. Similarly, MEK-1 kinase-mediated phosphorylation of the SMRT mammalian corepressor leads to the translocation of this factor from the nucleus to the cytoplasm in cell culture transfection assays. The functional homolog of this protein in flies, SMRTER, also shows active redistribution from the nucleus to the cytoplasm in response to a MAP kinase pathway, in this case mediated by EGFR/Sno/Ebi in the Drosophila eye. In both of these systems, regulated phosphorylation of SMRT/SMRTER results in dissociation of a repressor complex and derepression of target gene transcription (Gates, 2003).

These observations raise the possibility that the subcellular location of Rig could determine its regulatory function in different cell types. For example, by analogy with SMRT/SMRTER, loss of Rig from the nucleus of larval cells might disrupt a corepressor complex on specific promoters, leading to coordinate target gene derepression. This is consistent with the proposal that the ecdysone receptor exerts critical repressive functions during larval development. Alternatively, Rig protein in the cytoplasm may tether one or more nuclear receptors, preventing them from acting on their cognate target genes in the nucleus. This model is not favored, however, because antibody stains reveal an exclusively nuclear localization for EcR, USP and ßFTZ-F1 at the onset of metamorphosis. It is also interesting to note that Rig protein appears to localize to discrete regions within the nuclei of larval midgut cells that do not contain chromosomes while Rig co-localizes with the giant polytene chromosomes in larval salivary gland cells. Rig may thus exert some functions in the nucleus that are independent of chromatin binding. Further biochemical studies of Rig, including the identification of additional proteins that interact with this factor, should provide insights into the significance of the subcellular localization of Rig protein as well as a mechanistic understanding of how Rig contributes to ecdysone responses during Drosophila larval development (Gates, 2003).

The Drosophila nuclear receptor E75 contains heme and is gas responsive

Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still 'orphans'. The ligand binding pocket of the Drosophila nuclear receptor E75 is shown to contain a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed (Reinking, 2005).

Using electronic absorption and mass spectrometry, it has been shown that the Drosophila nuclear receptor E75 contains a single tightly associated heme prosthetic group. Thus, nuclear receptor LBDs can now be added to the limited repertoire of known heme binding motifs. All results here are consistent with a conventional heme-protein interaction mediated by a pair of coordinate bonds. Two highly conserved histidine residues are good candidates for the interacting residues (Reinking, 2005).

The results suggest three general ways in which E75-heme may function. (1) The necessity of heme for E75 LBD stability and the changes in expression levels brought about by supplemental heme suggest that E75 may function as a nuclear monitor of cellular heme levels. (2) The ability of E75-heme to switch between oxidized and reduced states and the effects of these states on CO, NO, and cofactor peptide binding suggest a possible role as a cellular redox sensor. (3) The ability of E75-heme to bind NO and CO, and for these gases to modulate cofactor binding and transcriptional activity, suggests a role in mediating NO and/or CO intercellular signaling. This is the first example of a nuclear receptor with a bipartite ligand binding system (Reinking, 2005).

Nuclear receptors were first characterized based on their ability to bind steroid hormones but are now known to bind a fairly diverse set of lipophilic molecules including fatty acids, phospholipids, retinoids, bile acids, farnesoids, and a range of xenobiotics. Like most other nuclear receptor ligands, heme is also a lipophilic molecule with polarized negative charges. Its molecular weight and solvent-excluded volume, although larger than most, are within the range of other known ligands. For example, one of the closest vertebrate homologs to E75, Peroxisome proliferator activator receptor gamma (PPARgamma), has a pocket that can easily accommodate a molecule of this size. Although heme is not dissimilar to other ligands in most physical attributes, an iron atom within a protoporphyrin ring brings an exciting new dimension to the potential molecular and physiological roles of nuclear receptor proteins (Reinking, 2005).

The tight interaction generally formed between heme molecules and their protein partners, also observed with E75, and the inability to detect apo-E75 suggest that E75 and heme are unlikely to associate and dissociate readily. For this to occur, dedicated cofactors would be required to shuttle the heme in and out of the LBD and to stabilize the LBD in the absence of heme. Although no evidence was found for such cofactors, it is possible that endogenous E75 is expressed in certain tissues or stages in which such cofactors exist. If, however, these cofactors are not present, or do not exist, heme would be required as a dedicated structural component. In the latter case, the levels of free heme in the cell would determine the levels of E75 that can accumulate. Either way, heme availability would determine the levels of active E75 in the cell. If E75 target genes include regulators of heme metabolism, these properties would make E75 an ideal regulator of heme homeostasis (Reinking, 2005).

Heme is required by proteins that control an enormous range of cellular and biological processes. Examples of these processes (and proteins) include energy transfer (mitochondrial cytochromes), lipid and drug metabolism (cytochrome P450s), heme metabolism (heme oxygenase [HO]), oxygen radical detection and removal (superoxide dismutases, catalase, NADPH hydroxylase), gas transport (hemoglobin, myoglobin), iron transport (HO-FE-ATP pump), neuronal differentiation, and behavior (guanylate cyclase, nitric-oxide synthase [NOS]) and circadian rhythm (HO, NPAS2). Interestingly, NOS and HO, the enzymes that produce NO and CO in the cell, are also heme-containing proteins (Reinking, 2005 and references therein).

Of the physiological processes listed above, particularly intriguing and relevant ones are lipid and xenobiotic metabolism. In vertebrates, most of the enzymes that regulate these reactions are cytochrome P450s, which typically use heme to transfer oxygen or hydroxyl groups to or from their substrates. Homeostasis between lipid absorption/production and secretion/breakdown is controlled by nuclear receptors, largely through transcriptional regulation of cytochrome P450 (cyp) genes. In turn, the substrates or products of the P450-regulated reactions are often ligands for the nuclear receptors that regulate expression of the respective P450 genes. This triangular relationship between transcriptional regulators, enzymes, and reaction products provides the necessary feedback for homeostasis. Given that a close link between heme and lipid metabolism is also well documented, the ability of a nuclear receptor to monitor levels of heme could provide a general mechanism for coordinating these processes (Reinking, 2005).

The iron center of E75 can reversibly switch between Fe(III) and Fe(II) oxidation states. Furthermore, the interaction with an HR3-derived peptide is selective for the Fe(II) oxidation state of E75-heme. This set of characteristics sets up the intriguing possibility of E75 acting as a direct redox sensor. An interesting example of this behavior is the reported heme-based redox sensor activity of Ec-DOS, a phosphodiesterase in E. coli. Enzymatic activity is modulated by an allosteric change in the heme-containing PAS domain. The heterodimerization and transcriptional activities of the NPAS2 and Bmal1 transcription factors, which regulate circadian rhythm, have also been shown to depend on redox potential in vitro. NPAS2 has two heme-containing PAS domains, which could provide the mechanism for redox detection. Ec-DOS and NPAS2 also function as gas sensors (Reinking, 2005).

In many heme bound proteins, the heme molecule serves as a cofactor or prosthetic group for the binding of diatomic gases. This is also the case for E75-heme, and NO and CO may serve as E75 ligand(s). Although small, these diatomic gases have been shown to bring about significant changes in protein structure. In heme-containing proteins such as hemoglobin, myoglobin, guanylate cyclase, CooA, and FixL, for example, the binding of O2, CO, or NO causes allosteric rearrangements that modulate protein multimerization, enzymatic activity, and/or the ability to interact with cofactors or target molecules (Reinking, 2005).

In the case of E75, peptide binding studies suggest that CO interferes with the ability of E75 to interact with the DHR3 AF2 motif. Although unable to confirm this by direct binding assays, the results with NO on E75 transcriptional activity suggest that NO may act similarly. This may be analogous to the case of soluble guanylate cyclase, where NO and CO binding both lead to functional activation, but to varying degrees. As with other nuclear receptors, proper placement of the DHR3 AF2 helix within its ligand binding domain is most likely required for DHR3 to bind transcriptional coactivators. If E75 were to sequester the DHR3 AF2 helix, then DHR3 activity would be compromised. By reversing the AF2-E75 interaction, NO and/or CO could then restore normal DHR3-coactivator interactions (Reinking, 2005).

NO and CO are being implicated in a rapidly growing list of signalling processes in vertebrates, and insects. Their ability to diffuse readily between and within cells, and their short half-lives, make them ideal intercellular signaling molecules. In many cases, they act together or in opposition. Examples of processes regulated by NO and CO include blood pressure, cell division, cell death, inflammation, metabolism, hypoxia, diurnal cycles, behavior, and memory. The primary source of NO in tissues is the enzyme NOS and for CO, heme oxygenase. In flies, mutation of the dnos gene results in embryonic lethality. Flies also contain a single heme oxygenase gene, but genetic and functional analyses of dHO are yet to be carried out (Reinking, 2005).

The fact that CO production is dependent upon heme as a substrate, that NOS and heme oxygenase have heme centers, and that they have related physiological functions poses an interesting set of coincidences. This and the convergence of heme with lipid synthesis and related processes, NO and CO gas binding and now E75 function, suggests that E75 may provide a unifying role in the regulation of these processes (Reinking, 2005).

Numerous studies have placed E75 genetically, transcriptionally, and functionally in the ecdysone response pathway, both upstream and downstream of the ecdysone receptor. Thus, E75 appears to play roles in both ecdysone production and response. Ecdysone-regulated processes during insect development include cuticle formation, molting, programmed cell death, neurogenesis, imaginal disk development, and oogenesis. The binding of E75 to heme suggests possible connections between these processes and heme metabolism or function. This could occur at a variety of different levels. Some intriguing possibilities include the regulation of hormone-synthesis pathways, oogenesis arrest, metabolism, and the control of circadian rhythm (Reinking, 2005).

An intimate functional triangle exists between nuclear receptor function, cytochrome P450 expression, and cytochrome P450 substrates. This relationship, taken together with the apparent need for E75 in ecdysone production and response, suggests the possibility that E75 could control ecdysteroid metabolism by regulating the transcription of key cytochrome P450 genes, or more globally by sensing the levels of available heme. The possible ability of E75 to regulate hormone synthesis, and its known role in controlling the progression of molts, metamorphosis, and oogenesis, also brings up other intriguing possible functions. One example might be the ability to monitor energy resources such as lipids and to coordinate these levels with developmental progression. More specifically, the close link between heme, NO, and lipid metabolism could be used to block or postpone molting, pupariation, or oogenesis when energy resources are low, and vice versa (Reinking, 2005).

Interestingly, in mosquitoes, oogenesis is halted prior to chorion deposition until the insect obtains a blood meal. Although the blood-meal components that release the oogenesis arrest have yet to be identified, one of the major components of the blood meal is heme removed from the metabolized hemoglobin. Taken together with the observations that one of the responses of the blood meal is a pulse of ecdysone synthesis and action and that in Drosophila, E75 functions in both a feed forward and downstream role in the ecdysone signaling pathway, E75 may play a key role in this response by responding to the ingested heme and inducing ecdysone synthesis (Reinking, 2005).

A number of possible links also exist between E75 function and a role in regulating circadian rhythms. (1) NO, CO, and heme appear to be important regulators of circadian oscillators. In vertebrates, 'light' and 'dark' inputs are thought to be converted to signals of NO and CO, which in turn modulate the phase and period of the circadian cycle. Heme biosynthesis is also reciprocally regulated by the circadian clock. (2) The closest homolog to E75 in vertebrates, Rev-Erbalpha, is a well-established regulator of circadian rhythm in mammals, also acting as a transcriptional repressor and competing with the nuclear receptor RORalpha, which is a transcriptional activator of the circadian regulator Bmal1. The interaction between Rev-Erbalpha and RORalpha is analogous to that of E75 and HR3, their closest insect homologs. (3) Each larval instar molt, as well as the onset of metamorphosis and adult eclosure, are coupled to the circadian clock. Molts between first and third instars occur every 24 hr, and metamorphosis and eclosure generally begin on the mornings of the fifth and ninth days. These events are regulated by rhythmic pulses of juvenile hormone and ecdysone, which are under E75 control. In insects that take longer to develop, such as Rodnius (21 days), ecdysone levels have been shown to oscillate daily, with levels highest at night. E75 may time these oscillating events indirectly by monitoring feeding activity (lipid intake) or through a more direct role in circadian rhythm (Reinking, 2005 and references therein).

An interesting implication of this study is that the orphan receptor, Rev-Erbalpha, may also bind heme and respond to diatomic gases. If it does not, compensating evolutionary steps may have been adopted to maintain the circuitry of E75-heme-regulated processes. It is also possible that other nuclear receptors may have adopted or conserved the capacity to bind heme and diatomic gases and to regulate corresponding developmental and physiological functions (Reinking, 2005).

PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. A Drosophila model has been developed of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, a drug screening strategy was designed based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, the PPARgamma agonist pioglitazone was found to be neuroprotective in Drosophila. This study shows that pioglitazone can rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS it was found that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, this study showed that the predicted Drosophila PPARγ homologs, E75 and E78 are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, a set of metabolites was identified that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, these data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, these data also identifies several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials (Joardar, 2014).

ALS is the third most common form of neurodegeneration following Alzheimer's and Parkinson's disease. Although Riluzole is approved for ALS patients, its benefits are marginal, and at this time, there are no known effective treatments for the disease. There have been several efforts to design therapeutics using the SOD1 mouse, the most commonly used animal model of ALS. However, despite promising preclinical results, these candidate drugs have been disappointing in humans. To address this significant issue, efforts are being made to develop other animal models of ALS that can be used not only to identify phenotypes and 'early biomarkers' of the disease but also will be useful in drug screens for therapeutic purposes. Previously work has generated a Drosophila model of ALS based on TDP-43, which recapitulates several aspects of the human disease including locomotor dysfunction and reduced lifespan Using this model, this study shows that the antidiabetic drug pioglitazone acts as a neuroprotectant for aspects of TDP-43 proteinopathy by activating the putative Drosophila PPARγ homologs E75 and E78. It was also shown that pioglitazone mitigates FUS but not SOD1-dependent toxicity in Drosophila, consistent with previous published work showing that distinct mechanisms are likely at work in the context of these different models of ALS. Interestingly, pioglitazone did not improve, and in some cases worsened, the lifespan of TDP-43-expressing flies, when administered either during development, or after 'disease onset', which is consistent with results from recent clinical trials. This apparent disconnect is consistent with the effects of pioglitazone on cellular metabolism. As described earlier, while pioglitazone treatment restored some metabolites altered owing to TDP-43 overexpression in motor neurons, others were unchanged or even worsened. This provides a potential explanation for why some phenotypes but not others are rescued by pioglitazone. Aside from the possibility that different drug concentrations may be needed, it remains unclear why pioglitazone is protective in mouse but not fly SOD1 models and, in retrospect, given the similarities between the effect of pioglitazone in Drosophila models of ALS and humans, the fly appears to be a more accurate predictor of clinical trial outcomes (Joardar, 2014).

It is tempting to speculate that the predictive power of the Drosophila model may lie in the tools that enable motor neuronal versus glial versus muscle-specific expression of the toxic TDP-43 protein. The current results show that pioglitazone mitigates neuronal and glial TDP-43-dependent toxicity but has no effect on the locomotor dysfunction caused by muscle-specific expression of TDP-43. This type of knowledge is easily obtainable in the fly model and can provide helpful information about cell autonomous versus non-autonomous effects as well as the efficacy of candidate drugs in different tissues of interest. While it was shown that pioglitazone reduces inflammation in the glia, its effects in neurons or muscles have not been studied in the mouse prior to human trial. The current results indicate that the protective effects of pioglitazone are specific to the nervous system and were not observed in muscles, at least within the limits of the experimental conditions (i.e., tissue-specific levels of expression and drug concentration). These findings suggest that future preclinical studies may benefit from testing candidate therapies in multiple disease models in which tissue specificity and several phenotypic outcomes are easily ascertained (Joardar, 2014).

Pioglitazone has been originally developed for the treatment of type 2 diabetes as PPARγ activation in the liver improves glucose metabolism systemically. In the nervous system, activation of the nuclear hormone receptor PPARγ has been shown to have anti-inflammatory and neuroprotective effects. In the current model, pioglitazone restored a rather limited set of metabolites altered in a TDP-43-dependent manner. Evidence was found evidence of altered glutamine/glutamate metabolism in TDPWT flies, as displayed by elevated levels of N-acetylglutamine, which is restored by pioglitazone. Excessive levels of extracellular glutamate in the central nervous system cause hyperexcitability of neurons, ultimately leading to their death. The glutamate transporter GLT1/EAAT2 plays a major role in maintaining extracellular glutamate levels below the excitotoxic concentrations by efficiently transporting this metabolite. Interestingly, astrocytic GLT1/EAAT2 gene is a target of PPARγ, leading to neuroprotection by increasing glutamate uptake. Furthermore, pyruvate, which is significantly high in both TDPWT and TDPG298S, shows a trend toward reduction upon pioglitazone treatment for TDPWT. Pyruvate is a central metabolite that lies at the junction of several intersecting cellular pathways including glucose and fatty acid metabolism. It is converted to oxaloacetate by the enzyme pyruvate carboxylase, which is a key step in lipogenesis. Interestingly, PPARγ, the target of pioglitazone, is a direct transcriptional modulator of the pyruvate carboxylase gene. Given the fact that ALS patients suffer from massive weight loss, this provides a possible explanation for the potential protective effects of pioglitazone through increased lipogenesis. Taken together, this metabolomics approach provides useful insights for understanding the molecular mechanisms underlying ALS pathophysiology. Interestingly, altered cellular metabolism has previously been implicated in ALS pathophysiology with patients exhibiting signs of hypermetabolism. Notably, the fly model also showed signs of hypermetabolism including an increase in pyruvate, a key metabolite linking glucose metabolism to the TCA cycle. Additionally, the ketone body GHB is reduced in the context of TDPWT, consistent with a clinical study showing that a ketogenic diet slowed ALS disease progression. Given the similarities between the metabolic profile of the Drosophila model and human samples, it will be interesting in the future, to design therapeutic approaches aimed at restoring these common metabolic changes using nutritional supplementation (Joardar, 2014).

In summary, these data show the potential of using the fly model of ALS as a rapid and efficacious system for drug screening in vivo. The results using FUS and SOD1 fly models of ALS indicate that pioglitazone is effective in mitigating some, but not all forms of the disease, which suggests that stratification of patient populations should be considered in future clinical trials. The primary endpoint tested in prior clinical trials, namely lifespan, was not improved by pioglitazone, which is consistent with the data in Drosophila. Although the results from the clinical trials have not shown much promise in ALS patients, the use of pioglitazone as a tool to dissect molecular mechanisms of the disease remains attractive. The metabolomic profiling with and without pioglitazone pinpoints pathways that could be targeted either by drugs or by diet modifications. Also, it is possible that chemical modifications of pioglitazone, which was optimized for adipose tissue, skeletal muscle and liver, are needed for increased efficacy in the nervous system. The protective effect of pioglitazone opens up avenues for designing small molecules with modifications around the basic structure of the drug, and testing their potential in vivo, in the fly model. Furthermore, clinical trials have not been stratified for TDP-43 pathology or mutations; thus, significant results may have been missed. The developmental and adult feeding experiments clearly demonstrate that locomotor function is improved by pioglitazone suggesting that despite its lack of effect on lifespan, PPARγ remains a molecular target with therapeutic potential, perhaps in combination with other strategies based on restoring the metabolic alterations caused by TDP-43 in the nervous system (Joardar, 2014).


DEVELOPMENTAL BIOLOGY

E75A is transcribed during each stage in the life cycle in bursts that accompany ecdysteroid peaks in the life cycle, as well as in late embryos in synchrony with the E74A early mRNA (Segraves, 1988; Thummel, 1990). The three E75 transcripts accumulate with distinct kinetics at the onset of metamorphosis, with peaks of E75A mRNA in late third instar larvae and late prepupae in synchrony with the ecdysteroid pulses, consistent with its direct induction by ecdysteroids (Andres, 1993; Huet, 1993; Karim, 1992; Segraves, 1990). E75B mRNA accumulates to peak levels in 2–4 hr prepupae, while E75C mRNA peaks in 10–12 hr prepupae (Bialecki, 2002).

In Drosophila, pulses of the steroid hormone ecdysone function as temporal signals that trigger the major postembryonic developmental transitions. The best characterized of these pulses activates a series of puffs in the polytene chromosomes as it triggers metamorphosis. A small set of early puffs is induced as a primary response to the hormone. These puffs encode regulatory proteins that both repress their own expression and activate a large set of late secondary response genes. Northern blot analysis of RNA isolated from staged animals and cultured organs has been used to study the transcription of three primary response regulatory genes, E75, BR-C and EcR. Remarkably, their patterns of transcription in late larvae can be defined in terms of two responses to different ecdysone concentrations. The class I transcripts (E74B and EcR) are induced in mid-third instar larvae in response to the low, but increasing, titer of ecdysone. As the hormone concentration peaks in late third instar larvae, these transcripts are repressed and the class II RNAs (E74A, E75A and E75B) are induced. The BR-C RNAs appear to have both class I and class II characteristics. These data demonstrate that the relatively simple profile of a hormone pulse contains critical temporal information that is transduced into waves of primary response regulatory gene activity (Karim, 1992).

The Drosophila E75 early gene has been isolated and two of its products, E75A and E75B, have been shown to be members of the steroid receptor superfamily. Antisera directed against A- and B-specific regions of the E75 proteins has been prepared. Antisera and a monoclonal antibody raised against E75A, the major larval protein product of the E75 gene, bind to discrete sites in native salivary gland chromosomes. These sites are closely correlated with early and late ecdysone responsive loci (Hill, 1993).

E75 expression and function during oogenesis

The ecdysone response hierarchy mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates (Buszczak, 1999).

During stage 10, the follicle cell expression of E75 becomes enriched in the dorsal anterior cells. This suggested that inputs in addition to ecdysone are needed to refine E75 expression. Previous work has shown that follicle cell polarity is established during mid- to late-oogenesis and depends on the interaction between Gurken and the Drosophila homolog of the mammalian EGF receptor (Egfr). To determine whether E75 expression is under control of the dorsoventral signaling pathway, ovarian E75 mRNA distribution was examined in dorsalized and ventralized mutant backgrounds. In fs(1)K10 mutants, mislocalization of Grk protein results in activation of Egfr in all anterior follicle cells surrounding the oocyte. In fs(1)K10 mutant egg chambers, E75 expression expands to a ring of anterior follicle cells surrounding the oocyte. Mutations in Egfr prevent signal transduction by the receptor and lead to the ventralization of the eggshell and embryo. In situ analysis indicates that stage 10 follicle cells overlying the oocyte in Egfr mutants no longer express E75. However, E75 expression in the nurse cells is unaffected. These experiments show that the Egfr signaling pathway regulates E75 expression in the dorsal follicle cells but not in the germline (Buszczak, 1999).

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).

Nutritional status affects 20-hydroxyecdysone concentration and progression of oogenesis in Drosophila melanogaster; E75 isoforms are involved in the decision to develop or die in oogenesis

The number of Drosophila egg chambers is controlled by the nutritional status of the female. There is a developmental checkpoint at stage 8, which is controlled by BR-C in the follicle cells along with ecdysteroid. During this period, developmental decision is made in each egg chamber to determine if it will develop or die. During nutritional shortage, inducing apoptosis in the nurse cells of stages 8 and 9 egg chambers reduces the number of egg chambers. Ecdysone response genes E75A and E75B are involved in inducing or suppressing apoptosis. It is thus possible that the E75 isoforms A and B are involved in the decision to develop or die in oogenesis. This study establishes part of the pathway by which ecdysone response genes control apoptosis of the nurse cells and hence select between degeneration or development of individual egg chambers at stages 8 and 9 (Terashima, 2005a).

Drosophila egg production depends upon the nutrition available to females. When food is in short supply, oogenesis is arrested and apoptosis of the nurse cells is induced at mid-oogenesis via a mechanism that is probably controlled by ecdysteroid hormone. Expression of some ecdysone-response genes is correlated with apoptosis of egg chambers. Moreover, ecdysteroid injection and application of juvenile hormone induces and suppresses the apoptosis, respectively. In this study, an investigation was carried out to see which tissues show increases in the concentration of ecdysteroids under nutritional shortage to begin to link together nutrient intake, hormone regulation and the choice between egg development or apoptosis made within egg chambers. Ecdysteroid levels in the whole body, ovaries and haemolymph samples were measured by RIA, and it was found that the concentration of ecdysteroid increased in all samples. This contributes to the idea that nutritional shortage leads to a rapid high ecdysteroid concentration within the fly and that the high concentration induces apoptosis. Low concentrations of ecdysteroid are essential for normal oogenesis. It is suggested there is threshold concentration in the egg chambers and that apoptosis at mid-oogenesis is induced when the ecdysteroid levels exceed the threshold. Starvation causes the ovary to retain the ecdysteroid it produces, thus enabling individual egg chambers to undergo apoptosis and thus control the number of eggs produced in relation to food intake (Terashima, 2005b).

The prothoracic glands, which are the principal source of ecdysone in the immature stages, are no longer present in adults. The egg chambers produce ecdysone, which, at least in some insects, accumulates in the oocyte. In the fat body, ecdysone is converted to 20E, the active hormone, and shade, which encodes 20-hydroxylase for converting ecdysone to 20E, is expressed in nurse cells and follicle cells in the ovary and fat body (Terashima, 2005b).

Ecdysteroid synthesis is affected by the nutritional status of the female, and ecdysteroids affect oogenesis in many insects. Egg production in mosquitoes is triggered by a blood meal. The digested products of the blood meal stimulate the brain to secrete egg development neurosecretory hormone (EDNH), which is also known as ovarian ecdysteroidogenic hormone (OEH). EDNH stimulates the ovary to synthesize ecdysteroids, which instruct the fat body cells to make vitellogenin for the oocytes. Vitellogenin is critical for egg production, thus without the blood meal there is no vitellogenin and no eggs, so to produce mature eggs ecdysteroids are essential. In contrast, nutritional shortage induces an increase in ecdysteroid concentration in Drosophila females, ecdysteroid concentration increases in Drosophila whole body, haemolymph and ovaries during starvation. Feeding suppresses the high ecdysteroid concentration that is induced by nutritional shortage (Terashima, 2005b).

Under starvation, apoptosis of nurse cells in stage-8 and -9 egg chambers is induced; 20E injection into the females under adequate nutrition also induces the apoptosis and JHA treatment of females under nutritional shortage suppresses this apoptosis. Presumably high ecdysteroid concentrations in the haemolymph and/or the ovary, which are induced by starvation, may induce the apoptosis of nurse cells in stage-8 and -9 egg chambers. However, ecdysteroid is indispensable to produce mature eggs in Drosophila. Oogenesis in ecd-1 mutants is arrested at mid-oogenesis, and germline clones of EcR mutations led to developmental arrest; egg chambers degenerated during mid-oogenesis in Drosophila. Presumably, there is an ecdysteroid threshold for inducing apoptosis of nurse cells at stages 8 and 9 and ecdysteroids induce normal development when below the threshold concentration and induce apoptosis of nurse cells at stages 8 and 9 when over the threshold. Starvation induces an increase in ecdysteroid concentration to above the threshold level in the haemolymph and the ovary through activation of the ecdysone synthesis pathway in the egg chamber. Ecdysteroid secretion from the ovary decreased following nutritional shortage. Thus, ecdysteroid secretion from the fat body or other ecdysteroid-synthesizing tissues must be stimulated to induce the high ecdysteroid concentration observed in haemolymph (Terashima, 2005b).

JHA suppresses the high ecdysteroid concentration that is induced by starvation. JH and JHA suppress ecdysone synthesis/secretion from the prothoracic glands in larvae of Maduca sexta. It is likely that JHA suppression decreases the high ecdysteroid concentration in the ovary that induces apoptosis of nurse cells in stage-8 and -9 egg chambers under starvation, and therefore JHA treatment retains minimal ecdysteroid levels needed for inducing normal oogenesis (Terashima, 2005b).

There is a developmental checkpoint at stage 8 of oogenesis. YP synthesis commences at stage 8 and YP is accumulated during development into mature eggs. Drosophila egg chambers normally transit through stages 8 and 9 during a 6-h period, but starvation induced an accumulation of stage-8 and -9 egg chambers in Drosophila oogenesis. The number of stage-8 egg chambers is increased during a 5-12-h period after starvation starts, but the number of stage-9 egg chambers does not increase for 0-12 h after starvation started. This means that oogenesis progresses from stage 7 to 8, but does not progress from stage 8 to 9 and then to 10 under nutritional shortage. When 20E is injected into the fed flies, the accumulation of stage-8 chambers is not seen; therefore this arrest of oogenesis at stage 8 was not caused by the increasing 20E concentration in haemolymph and ovary. Perhaps starvation signals induce the arrest of oogenesis at stages 8 and 9 directly, or they could inhibit YP uptake. Some nutrient- and stress-response genes exhibit different expression patterns in the ovaries of females under adequate nutrition and starvation. It is suggested that the genes which respond directly to stress and nutrients interact with the ecdysone-synthesis pathway, resulting in the induction of apoptosis of nurse cells in stage-8 and -9 egg chambers through activation of BR-C Z2, Z3 and E75A expression in the follicle cells. Other genes could have altered their expression levels, so as to arrest oogenesis at stages 8 and 9 and to check the developmental status of the egg chamber. As a result, the decision is made to develop into a mature egg or undergo apoptosis at stages 8 and 9. The arrest in the progression of oogenesis at stages 8 and 9 is independent of increasing ecdysteroid levels (Terashima, 2005b).

Starvation signals are needed to activate a number of pathways to adjust the rate of egg production in Drosophila. These pathways could be classified into two groups: one to stimulate ecdysone synthesis in the follicle cells and/or nurse cells to activate the apoptosis pathway, including BR-C Z2, Z3 and E75A expression in the follicle cells, and another one to interact with and participate in the developmental checkpoint, giving rise to an arrest in oogenesis at stage 8 under nutritional shortage. A possible scheme is presented for the regulation of oogenesis related to nutrition in Drosophila. It is likely that starvation signals from the gut activate ecdysteroid synthesis in the ovary in Drosophila under starvation. Ecdysteroid is then accumulated in the egg chamber by decreasing 20E secretion from the ovary, and the fat body secretes 20E to haemolymph. It is suggested that there are two thresholds of 20E concentration in Drosophila ovary -- one is the concentration for normal oogenesis and the other is the concentration for inducing apoptosis -- and that starvation elevates the ecdysone levels in some egg chambers over the threshold that leads to apoptosis (Terashima, 2005b).


EFFECTS OF MUTATION

Imprecise excision of P elements was used to generate small deletions of sequences specific to either E75A or E75B, and to create a larger deletion of common region sequences shared by all three E75 isoforms. E75A81 is a 1.8 kb deletion that removes the E75A transcription start site along with the 5'-untranslated region and 143 bp of protein-coding sequence. E75D1 is an ~3 kb deletion that removes the E75B transcription start site along with most of the first E75B exon. E75D51 is an ~30 kb deletion that removes the first exon of E75B as well as the adjacent exon, shared by all three E75 isoforms, that encodes the second zinc finger of the DNA binding domain. In homozygotes and heteroallelic combinations, E75D51 and E75e213, a putative null EMS-induced point mutation in the E75 common region (Buszczak, 1999; Segraves, 1988), led to similar lethal phases and phenotypes, arguing that the E75D51 mutation inactivates all E75 functions. E75x37 is an ~60 kb g irradiation-induced deletion that removes the E75C transcription start site as well as ~10 kb of the downstream primary transcript (Segraves, 1988). E75x37 fails to complement another E75C allele, E75e273, which is an EMS-induced 63 bp deletion that removes the E75C splice donor (Segraves, 1988). E75x37 and E75e273 generate identical lethal phenotypes when maintained over a deletion for the E75 locus, providing further evidence that E75C is the only essential E75 function affected by the x37 deficiency (Bialecki, 2002).

Similar lethal phases and phenotypes were observed for E75A81 and E75x37 mutants when examined as either homozygotes or over the E75D51 common region deficiency, suggesting that they are null alleles with respect to the affected isoform. All studies described here use an isoform-specific E75 mutation over the E75D51 common region deficiency. For simplicity, E75D51 is referred to as the deficiency (Df) for the E75 locus, E75A81/Df as E75A mutants, E75D1/Df as E75B mutants, and E75x37/Df as E75C mutants. mRNA corresponding to the affected E75 isoform was undetectable by Northern blot hybridization in each of these genotypes, consistent with the molecular nature of these lesions and provides further evidence that they are null alleles (Bialecki, 2002).

Crosses were set up between control ry506/TM6B Ubi-GFP, E75D1/TM6B Ubi-GFP, or E75x37/TM6B Ubi-GFP animals and the deficiency stock E75D51/TM6B Ubi-GFP to test for embryonic lethality among the offspring. From the ry506 control cross, 26% of the first instar larvae did not express GFP, indicating no significant embryonic lethality. Similar results were obtained from the crosses with E75B mutants (23%) and E75C mutants (25%) (Bialecki, 2002).

Analysis of E75B and E75C mutants

E75B and E75C mutant first instar larvae were collected at hatching and examined at regular intervals for phenotypes and lethality during later stages of development. E75B mutants are viable and fertile, with no detectable phenotypes. In contrast, E75C mutants display lethality during both the pharate adult and adult stages. Approximately 33% of E75C mutants die as pharate adults, with normal adult pigmentation and fully developed appendages. The remaining E75C mutants eclose and are severely uncoordinated, displaying difficulty in walking and an inability to fly. These animals die within a week following eclosion. E75C mutant adults appear morphologically normal with the exception of black spots that cover about one quarter of the surface of the eye (Bialecki, 2002).

E75B and E75C are both induced by ecdysteroids at the onset of metamorphosis, suggesting that they may function during this stage in development (Huet, 1993; Karim, 1992). Gain-of-function studies have also implicated E75B in contributing to the timing of ßFTZ-F1 expression in mid-prepupae (White, 1997). The temporal profiles of ecdysteroid-regulated gene transcription were examined in E75B and E75C mutant late larvae and prepupae. Total RNA was isolated from control, E75B, and E75C mutant late third instar larvae staged at -18, -8, and -4 hr relative to puparium formation, as well as from prepupae staged at 2 hr intervals after puparium formation. These RNA samples were analyzed by Northern blot hybridization using radiolabeled probes designed to detect 16 ecdysteroid-regulated transcripts: EcR, BR-C, E74A, E74B, E75A, E75B, E75C, E78B, DHR3, Imp-E1, Fbp-1, Sgs-4, L71-1, L71-3, ßFTZ-F1, and Edg84A (Bialecki, 2002).

All of the tested transcription units, with the exception of E75B, are expressed normally in E75B mutant larvae and prepupae. Focus was placed on the temporal profile of ßFTZ-F1 expression in this mutant, and several independent Northern blots were prepared, one of which utilized prepupae staged at 30 min intervals. No reproducible effects on ßFTZ-F1 expression, however, could be detected. It is thus concluded that E75B is not required for the appropriate timing of ßFTZ-F1 transcription during prepupal development (Bialecki, 2002).

The tested transcription units are also expressed normally in E75C mutants at the onset of metamorphosis, with four exceptions. E75B mRNA normally peaks in abundance at 2 hr after puparium formation and is reinduced ~8 hr later, in response to the prepupal ecdysteroid pulse (Huet, 1993; Karim, 1992). This reinduction is submaximal in E75C mutants. Interestingly, the E75C mutation has a similar effect on Fbp-1, L71-1, and L71-3 transcription at this stage in development. Fbp-1 encodes a larval serum protein receptor that is induced by ecdysteroids in the fat body of mid-third instar larvae, and L71-1 and L71-3 are late ecdysteroid-inducible genes that are specifically expressed in prepupal salivary glands. These genes are normally downregulated in early pupae, ~14 hr after puparium formation. In E75C mutants, however, this repression occurs prematurely, ~10 hr after puparium formation. It is at this time that E75C mRNA levels peak in abundance in wild-type animals (Andres, 1993; Karim, 1992), indicating that E75C is normally required to maintain the expression of these genes through the prepupal-pupal transition (Bialecki, 2002).

E75 deficiency causes larval lethality while E75A mutants die throughout development

To assess possible embryonic lethality, E75A81/TM6B Ubi-GFP animals were crossed to the deficiency stock E75D51/TM6B Ubi-GFP. From this cross, 20% of the offspring hatched as first instar larvae that did not express GFP. Similarly, 20% of the offspring from E75D51/TM6B Ubi-GFP adults hatched as first instar larvae that did not express GFP. These numbers are lower than the 26% observed in the ry506 control cross, indicating some embryonic lethality. Embryonic lethality in E75 common region mutants has been shown to be associated with abnormal midgut morphogenesis (Bilder, 1995) as well as head involution defects (Bialecki, 2002).

E75A and E75D51 common region mutant first instar larvae were collected at hatching and examined at regular intervals for lethality during later stages of development. E75D51 common region mutants remain as first instar larvae for over a week before dying without any detectable morphological abnormalities. Some E75A mutants also arrest development as first instar larvae, although lethality is also observed at most other stages in the life cycle including second instar larvae, third instar larvae, early pupae, and pharate adults. Developmental delays, developmental arrests, and molting defects were observed in these animals. Subsets of first and second instar mutant larvae never molt to the next instar, surviving for up to a week before dying. Those that molt do so up to 12 hr late, while some die at the molt. These animals often have malformed mouthhooks or two sets of mouthhooks, indicative of a molting defect. E75A mutant pharate adults display no detectable morphological defects, but fail to eclose (Bialecki, 2002).

Mutants in E75A may be deficient in production of ecdysteroid

Developmental delays, developmental arrests, and molting defects seen in E75A mutants are characteristic of an ecdysteroid deficiency. Approximately 20% of E75A mutant second instar larvae display a heterochronic phenotype in which they live for several days beyond the time when they should have molted to the third instar, and then pupariate (denoted as L2 prepupae). These delayed second instar larvae continue to eat and grow, exceeding the size of wild-type second instar larvae, approaching the size of a wild-type late third instar larva. They begin to pupariate ~88 hr after the first-to-second instar larval molt, forming what will be referred to hereafter as L2 prepupae (Bialecki, 2002).

The L2 prepupae appear to be derived from second instar larvae based on three criteria: (1) L2 prepupal anterior spiracles do not evert and consist of a single club-shaped spiracular opening, identical to that of second instar larvae, in contrast to the everted spiracular papillae characteristic of a wild-type prepupa derived from a third instar larva; (2) mouthhooks dissected from L2 prepupae, although malformed to varying degrees, are more similar to wild-type second instar larval mouthhooks, both in size and tooth structure, than to wild-type third instar larval mouthhooks; (3) no ejected mouthhooks or shed cuticle could be found in the media of E75A mutant L2 prepupae by the time they pupariated. Remarkably, about 20% of the L2 prepupae develop to the pupal stage as evidenced by head eversion and leg and wing extension, indicating that these animals respond in a relatively normal manner to the prepupal ecdysteroid pulse (Bialecki, 2002).

The majority of E75A mutants display defects during the second larval instar. In an initial effort to understand the molecular basis of these defects, total RNA was isolated from staged control and E75A mutant second instar larvae and analyzed by Northern blot hybridization using probes to detect the expression of eight genes: E74, ßFTZ-F1, EcR, usp, dare, dib, Lcp-b, and rp49. Focus was placed on three regulatory genes that are expressed during the second instar: EcR, E74, and ßFTZ-F1. These genes are coordinately induced in wild-type second instar larvae 15–18 hr after the molt. Expression of E74 and ßFTZ-F1 is significantly affected in E75A mutant second instar larvae. E74B mRNA can be detected throughout the time course, accumulating to higher levels at later times, while E74A mRNA accumulation is both significantly reduced and delayed. ßFTZ-F1 mRNA is also significantly reduced in E75A mutant second instar larvae. The effects on EcR mRNA accumulation, however, are relatively minor, with EcR failing to be repressed at later times. These blots were also hybridized to detect usp mRNA, which is unaffected by the E75A mutation. The expression of EcR and usp mRNA in E75A mutant second instar larvae suggests that the defects in this mutant cannot be attributed to a reduced level of ecdysteroid receptor at this stage in development (Bialecki, 2002).

The pattern of E74 transcription seen in E75A mutants is similar to that seen in third instar larval organs treated with a low concentration of 20E. This observation raises the possibility that E75A mutant second instar larvae might be ecdysteroid deficient. This proposal is further supported by the lethal phenotypes of E75A mutants that resemble those seen in ecdysteroid-deficient mutants. Accordingly, the expression was examined of two genes that are known to be directly involved in the ecdysteroidogenic pathway in Drosophila: dare and disembodied (dib). dare encodes the Drosophila ortholog of adrenodoxin reductase, a mammalian enzyme that plays a central role in vertebrate steroid hormone biosynthesis by transferring electrons to all known mitochondrial cytochrome P450s. Genetic studies of dare mutants suggest that this gene plays a similar role in Drosophila ecdysteroid biosynthesis. dib encodes a presumptive target for dare action, a cytochrome P450 that is essential for ecdysteroid biosynthesis in Drosophila. Both dare and dib mRNA, however, are expressed throughout the second larval instar and appear to be unaffected by the E75A mutation. Similar results were obtained with separate collections of animals and with poly(A)+ RNA from E75A mutant second instar larvae (Bialecki, 2002).

Lcp-b expression was also examined in E75A mutant second instar larvae. This gene encodes a larval cuticle protein that is induced during the latter half of the second instar. Lcp-b mRNA is upregulated at 18 hr after the molt, in synchrony with EcR, E74A, and ßFTZ-F1, and accumulates to higher levels throughout the second instar, consistent with earlier results (Charles, 1998). Lcp-b transcription is slightly delayed and reduced in E75A mutant larvae but otherwise expressed normally, indicating that these animals can faithfully express a marker for the second instar stage (Bialecki, 2002).

A subset of E75A mutant second instar larvae fails to molt to the third instar and pupariate, forming L2 prepupae. To determine whether these animals execute genetic programs specific to later stages of development, the patterns of E74, Sgs-4, and Fbp-1 transcription were examined in E75A mutant second instar larvae. These ecdysteroid-inducible genes are normally expressed during the second half of third larval instar and thus provide molecular markers for animals that are progressing toward the onset of metamorphosis (Bialecki, 2002).

Total RNA was collected from control third instar larvae staged from 24 to 44 hr after the second-to-third instar molt, and E75A mutant second instar larvae staged from 48 to 88 hr after the first-to-second instar molt. Control larvae begin to pupariate at ~48 hr after the second-to-third instar molt, while the majority of E75A mutant second instar larvae begin to pupariate after 88 hr. RNA extracted from both sets of animals was analyzed by Northern blot hybridization (Bialecki, 2002).

E74B is expressed throughout the second half of the third larval instar in wild-type animals, and begins to be repressed as E74A mRNA is induced by the high-titer late larval ecdysteroid pulse. A similar pattern is seen in E75A mutant second instar larvae, although E74A induction is significantly delayed and reduced, detectable at 84 hr after the first-to-second instar molt. Both Sgs-4 and Fbp-1 are also induced in E75A mutant second instar larvae, although their normally coordinate induction is disrupted, with Fbp-1 induced 56–64 hr after the molt, and Sgs-4 induced 76–84 hr after the molt in mutant animals. The expression of Sgs-4 and Fbp-1 in E75A mutant second instar larvae indicates that they are capable of inducing genetic programs specific to the third instar stage (Bialecki, 2002).

Approximately 20% of E75A mutant L2 prepupae undergo head eversion, forming pupae with elongated legs and wings. In an effort to determine whether these developmental changes reflect normal transcriptional responses to ecdysteroids, the expression was examined of two key ecdysteroid-regulated genes that respond to dynamic changes in ecdysteroid titer in prepupae: E74 and ßFTZ-F1. E74A is expressed in newly formed prepupae and is repressed as E74B is induced by the rising ecdysteroid titer during prepupal development. E74B is then repressed as E74A is induced by the prepupal ecdysteroid pulse, followed by rapid repression of E74A and reinduction of E74B. In contrast, ßFTZ-F1 expression is restricted to the interval of low-ecdysteroid titer in midprepupae (Bialecki, 2002).

Total RNA was isolated from control prepupae and pupae staged at 2 hr intervals from 0 to 14 hr after puparium formation, as well as from staged E75A mutant L2 prepupae, and analyzed by Northern blot hybridization to detect E74 and ßFTZ-F1 transcription. Remarkably, the L2 prepupae display relatively normal patterns of E74 and ßFTZ-F1 expression, with a delay of ~2 hr in E74A mRNA accumulation. This observation suggests that the E75A mutants that pupariate from the second instar can execute appropriate changes in ecdysteroid titer during the onset of metamorphosis (Bialecki, 2002).

The conclusion that developmental delays, developmental arrests, and molting defects seen in E75A mutants are characteristic of an ecdysteroid deficiency is further supported by the pattern of E74 transcription in E75A mutant second instar larvae. In order to test this possibility, attempts were made to rescue the second-to-third instar larval molt by feeding ecdysteroids to E75A mutant second instar larvae. Second instar larvae staged 12–18 hr after the molt were transferred to yeast paste supplemented with either no hormone, 0.33 mg/ml 20E, 10 mg/ml 20E, or 0.66 mg/ml ecdysone. The animals were transferred to regular yeast paste after 6 hr in order to simulate the hormone pulse that triggers the molt, and then scored for animals that molted to the third instar after 18 hr. Almost all E75A mutant second instar larvae that were maintained on food without ecdysteroids failed to molt, either staying as second instar larvae or forming L2 prepupae. In contrast, the majority of larvae fed either ecdysone or 20E molted properly. Interestingly, about half of the mutant animals that molted to the third instar continued to develop to pupal and pharate adult stages, consistent with a critical role for E75A during the second larval instar (Bialecki, 2002).

These studies of E75A mutant second instar larvae suggest that they could have a reduced ecdysteroid titer at this stage of development. As a direct test of this hypothesis, the ecdysteroid titer was measured in these animals using an enzyme immunoassay (EIA). Control and E75A mutant second instar larvae were collected at 0–6, 6–12, 12–18, and 18–24 hr after the first-to-second instar larval molt. Organic extracts were prepared from these animals and the ecdysteroid titer was measured by EIA using a monoclonal antibody directed against 20E. Wild-type larvae show a peak of 20E at 6–12 hr after the molt, with the titer decreasing toward the end of the instar. In contrast, this peak is eliminated in E75A mutants, which also show a reduced basal level of 20E at all stages. To confirm these results, the EIA was repeated using a second set of control and E75A mutant larvae, collected at 0–12, 12–24, and 24–36 hr after the first-to-second instar larval molt. Similar results were obtained from these animals indicating that the ecdysteroid peak in E75A mutants is not delayed until 24–36 hr after the molt (Bialecki, 2002).

E75 and migdut development

The Drosophila midgut is an excellent system for studying the cell migration, cell-cell communication, and morphogenetic events that occur in organ formation. Genes representative of regulatory gene families common to all animals, including homeotic, TGF beta, and Wnt genes, play roles in midgut development. To find additional regulators of midgut morphogenesis, a set of genomic deficiencies was screened for midgut phenotypes. Fifteen genomic intervals necessary for proper midgut morphogenesis were identified, three contain genes already known to act in the midgut. Three other genomic regions are required for formation of the endoderm or visceral mesoderm components of the midgut. Nine regions are required for proper formation of the midgut constrictions. The E75 ecdysone-induced gene, which encodes a nuclear receptor superfamily member, is the relevant gene in one region and is essential fo proper formation of midgut constrictions. E75 acts downstream of the previously known constriction regulators or in parallel. Temporal hormonal control may therefore work in conjunction with spatial regulation by the homeotic genes in midgut development. Another genomic region is required to activate transcription of the homeotic genes Antp and Scr specifically in visceral mesoderm. The genomic regions identified by this screen provide a map to novel midgut development regulators (Bilder, 1995).

Steroid signaling within Drosophila ovarian epithelial cells sex-specifically modulates early germ cell development and meiotic entry

Drosophila adult females but not males contain high levels of the steroid hormone ecdysone, however, the roles played by steroid signaling during Drosophila gametogenesis remain poorly understood. Drosophila germ cells in both sexes initially follow a similar pathway. After germline stem cells are established, their daughters form interconnected cysts surrounded by somatic escort (female) or cyst (male) cells and enter meiosis. Subsequently, female cysts acquire a new covering of somatic cells to form follicles. Knocking down expression of the heterodimeric ecdysteroid receptor (EcR/Usp) or the E75 early response gene in escort cells disrupts 16-cell cyst production, meiotic entry and follicle formation. Escort cells lose their squamous morphology and unsheath germ cells. By contrast, disrupting ecdysone signaling in males does not perturb cyst development or ensheathment. Thus, sex-specific steroid signaling is essential for female germ cell development at the time male and female pathways diverge. These results suggest that steroid signaling plays an important sex-specific role in early germ cell development in Drosophila, a strategy that may be conserved in mammals (Morris, 2012).

These studies show that ecdysone signaling promotes multiple, fundamental steps of early oogenesis. Steroid signaling maintains the structure of the GSC niche and allows somatic niche cells to support a normal rather than a reduced number of GSCs. Subsequently, this pathway promotes 16-cell cyst production, meiotic entry and follicle formation. In contrast, male germ cell development lacks a steroid signaling requirement. Despite the fact that male somatic cyst cells interact with developing male germ cells in a very similar manner as in the ovary, and that male cysts form and enter meiosis like their female counterparts, disrupting steroid production or steroid pathway genes for eight days in these cells caused no detectable effect (Morris, 2012).

Ecdysone signaling was previously reported to be essential for initiating cystoblast development and for cell adhesivity. Germaria from flies in which signaling was reduced using similar methods to those applied in this study accumulated excess single-spectrosome-containing germ cells (cystoblasts). In contrast, no extra cystoblasts were seen unless knock down flies were followed beyond 8 days. The appearance of extra cystoblasts after prolonged gene knock down correlated with extensive alterations in the normal structure of the GSC niche and anterior germarium. The blockade in cystoblast specification/differentiation is therefore likely to be secondary to changes in somatic support cell shape and function, which are required to limit the range of the BMP signals repressing germ cell differentiation. Consequently, it is believed that ecdysone signaling directly affects the processes described here, but is only secondarily involved in cystoblast differentiation (Morris, 2012).

The formation of 16-cell cysts and entry into meiosis are closely linked. Shortly after completing synchronous mitoses that generate a new 16-cell cyst, all the germ cells enter the first meiosis-specific process, pre-meiotic S phase. The strong reduction in meiotic, 16-cell cyst formation that was observed when ecdysone signaling is reduced, suggests that hormones control meiotic entry during Drosophila oogenesis. Meiosis in many lower organisms is induced by nutrient limitation and modulated by nutrient-sensitive pathways Ecdysone signals may help determine when cysts have been starved sufficiently to enter meiosis, much as they assess nutrient sufficiency at other decision points (Morris, 2012).

If steroid signaling in the ovarian soma acts to mediate the extraordinary metabolic demands of female gamete production, then the absence of a male requirement is not surprising. The metabolic demands of egg production are immense, unlike those of sperm production. Thus, decisions affecting oocyte progression may have evolved to employ conserved mechanisms also used during life stage transitions such as dauer formation in C. elegans or the larval/pupal transition. This fundamental difference between male and female gametogenesis may apply to a wide range of organisms and might explain why sex-specific steroid signaling is a common aspect of gametogenesis (Morris, 2012).

Steroid hormone signaling plays a major role in mammalian sex determination and gametogenesis. Transcriptional changes controlled by the Y chromosome-linked SRY gene and hormonal differences dependent on the Sf1 nuclear receptor begin to orchestrate divergent germ cell developmental fates in the bipotential mouse gonad. At this stage, germ cells in both the both male and female gonad are engaged in cyst formation. In females, cysts are completed and enter meiosis while in the testis cyst formation and gamete development arrests. Whether estrogen mediates cyst completion and meiotic entry in female mice in a manner similar to the role of ecdysone in Drosophila remains an interesting question. Squamous, pre-granulosa cells surround mouse germline cysts at the time of follicle formation, and treatment of pregnant animals with estrogen or progesterone enhances the production of multi-oocyte follicles. This raises the possibility that steroid signaling also plays a conserved role during mammalian follicle formation (Morris, 2012).


EVOLUTIONARY HOMOLOGS

The ecdysone-inducible E75 gene responsible for the 75B puff of Drosophila melanogaster encodes a family of proteins which are members of the steroid receptor superfamily. These proteins are believed to be involved in the regulation of ecdysone response. In order to investigate the evolutionary conservation of E75, the E75 gene of Manduca sexta has been identified. The putative DNA binding, hormone binding and amino and carboxy terminal flanking domains of Drosophila E75 gene are conserved in Manduca E75. However, due to a relative reduction in intron size and number and the absence of homopolymeric amino acid repeats, the E75 B transcription unit and protein are considerably smaller in M. sexta than in D. melanogaster. These findings have implications for the identification of critical structural features of E75 and also suggest that E75 has a conserved function and a shared ligand in Lepidoptera (Segraves, 1993).

Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, a genomic clone from Galleria mellonella has been isolated that reveals 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encodes a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence shows 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone does not encode the N-terminal zinc finger, but includes a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicate that the Galleria gene E75 encodes at least two isoforms, GmE75A and GmE75B, that differ in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlate closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appear in high amounts within 4 h of the ecdysteroid rise, then decline concurrently with the hormone titer decline. At the time of pupal ecdysis, there is another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA is induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment (Jindra, 1994).

Cultured IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily has shown that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, it is concluded that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes (Palli, 1995).

A cDNA of the spruce budworm, Choristoneura fumiferana, that shows high amino acid similarity with the deduced amino acid sequences of E75 cDNAs cloned from Manduca sexta, Galleria melonella, and Drosophila melanogaster, has been cloned and characterized. The longest open reading frame of this cDNA has 690 codons and its deduced amino acid sequence has all five domains typical of a steroid hormone nuclear receptor. The deduced amino acid sequence of this cDNA shows the highest identity with the deduced amino acid sequence of E75A cDNAs cloned from M. sexta, G. melonella, and D. melanogaster, and is therefore named Choristoneura hormone receptor 75A (CHR75A). The CHR75A cDNA probe detects a 2.6 kb mRNA that is abundant at the time of the ecdysteroid peaks during molting in the embryonic, larval and pupal stages. In the sixth instar larvae, CHR75 mRNA is detected in the epidermis, fat body and midgut, and maximum expression is observed during the prepupal peak of ecdysteroids in the hemolymph. CHR75 mRNA is induced in ecdysone treated CF-203 cells and in the midgut, fat body and epidermis of larvae that are fed the non-steroidal ecdysteroid agonist, RH-5992. In vitro transcription and translation of the CHR75A cDNA yields a 79 kDa protein that binds to the retinoic acid receptor related orphan receptor response element (Palli, 1997).

Degenerate primers were derived from the amino acid sequence in the DNA binding domain of the Drosophila ecdysone receptor (DmEcR). Several partial cDNAs were amplified from the shrimp epidermis by reverse transcription polymerase chain reaction (RT-PCR). One of these fragments shows the highest amino acid sequence homology to the insect ecdysone inducible gene E75. This partial cDNA was used as a probe to screen the swimming leg cDNA library of the shrimp, Metapenaeus ensis. A 3.6 kb cDNA clone was obtained. The longest open reading frame of this cDNA consists of 606 amino acids and its deduced amino acid sequence has all five domains typical of a nuclear receptor. The putative polyadenylation signal is located at about 400 bp 3' to the stop signal. The deduced amino acid sequence of this cDNA shows the highest identity to that of the E75A reported in Manduca sexta, Galleria melonella, Drosophila melanogaster, and Choristoneura fumiferana. Based on the amino acid sequence comparison, the shrimp nuclear receptor is considered the insect homolog of E75A. Northern blot analysis shows that the shrimp E75 is expressed in the epidermis, eyestalk and the nerve cord of the pre-molt shrimp. Moreover, E75 transcripts can be detected in the epidermal tissues of early pre-molt shrimp by in situ hybridization. To determine whether the shrimp could also express other E75s like the insects, 5' end RACE and RT-PCR were performed on epidermal cDNA of a single shrimp. Subcloning and DNA sequence determination of the PCR products confirmed the presence of two other forms of E75 (tentatively called E75C and E75D) in shrimp. By RT-PCR, different levels of E75 expression can be detected in the epidermis, nerve cord and the eyestalk of early pre-molt shrimp. In addition to the different levels of expression of the shrimp E75s in the epidermis, the pattern of their expression is also different during the molting cycle. This is the first report on the cloning of a shrimp nuclear receptor superfamily member (Chan, 1998).

The homolog of the ecdysteroid-induced transcription factor E75A in Drosophila melanogaster was cloned from the tobacco hornworm, Manduca sexta, and its developmental expression and hormonal regulation were analyzed. Both E75A and E75B mRNAs are found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20E in vitro causes the rapid, transient induction of E75A RNA with a peak at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) shows a 10-fold higher sensitivity to 20E. The presence of the protein synthesis inhibitor anisomycin has no effect on the induction but prevents the decline, indicating that E75A RNA is directly induced by 20E, but its down-regulation depends on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, causes an increased accumulation of E75A RNA throughout the culture period although the temporal pattern is unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis (Zhou, 1998).

The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes thatencode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, ecdysone-regulated gene expression has been investigated in vitellogenic mosquito ovaries and fat bodies. The homolog of the Drosophila E75 early ecdysone inducible gene has been identified in the yellow fever mosquito Aedes aegypti. As in Drosophila, the mosquito homolog, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses (Pierceall, 1999).

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, the apoptosis of the prothoracic gland was analyzed of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, prothoracic gland degeneration was shown to be effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species (Mané-Padrós, 2010).

In Drosophila, a similar ecdysteroid-dependent cascade directs the stage-specific destruction of the salivary glands. In this insect, however, the cascade converges in the induction of rpr and hid, and the consequent massive caspase activation and cell death. Interestingly, RPR and HID homologous proteins have not been reported in any insect outside Drosophila and closely related species, suggesting that although the role of 20E as inducer of cell death is conserved, the mechanisms by which the hormone controls such process would be different in hemimetabolous insects (Mané-Padrós, 2010).

This study has demonstrated that BgFTZ-F1 is a critical factor to specifically induce the degeneration of the prothoracic gland of B. germanica after the imaginal molt. Several observations support this: (1) the prothoracic gland of BgFTZ-F1 knockdown adults failed to degenerate; (2) ectopic expression of BgFTZ-F1 in mid-last nymphal instar, mediated by the reduction of BgE75, prematurely induced the degeneration of the prothoracic gland; (3) the prothoracic gland of last instar nymphs that had been simultaneously knockdown for BgE75 and BgFTZ-F1, thus preventing the ectopic expression of BgFTZ-F1 during mid-nymphal instar, also failed to degenerate; (4) the expression of BgFTZ-F1 in the prothoracic gland during the imaginal molt is significantly higher (12-fold) than during nymphal transitions, and its expression is maintained during the first days of the adult stage until the onset of prothoracic gland degeneration (Mané-Padrós, 2010).

The nuclear receptor FTZ-F1 has also been implicated in the apoptotic response in Drosophila. In this insect, βFTZ-F1 acts as a transcriptional competence factor that provides the stage-specificity for salivary gland degeneration at the onset of the pupal development. Moreover, its activity is required for the destruction of the cytoplasm, nuclear DNA fragmentation and controlling caspase levels in salivary gland cells (Mané-Padrós, 2010).

In addition, it was also demonstrated that the pro-apoptotic effect of BgFTZ-F1 is restricted to the last nymphal instar due to the anti-apoptotic effect exerted by JH before the last nymphal instar. Although JH regulates a number of developmental events in insects, its molecular mechanism of action still remains a matter of debate. The death inhibitor effect of JH has been also described in relation to the midgut remodelling that occurs during metamorphosis of the mosquito A. aegypti and the moth H. virescens. Recently, it has been also shown that the larval fat body of JH-deficient Drosophila undergoes premature degeneration and that this response can be prevented by the application of methoprene. Interestingly, however, the anti-apoptotic effect of methoprene in Drosophila was based on the inhibition of two bHLH-PAS transcription factors involved in JH action, Methoprene-tolerant and Germ-cell expressed, but was not related, in contrast to B. germanica, to the suppression of the ecdysteroid-dependent transcriptional cascade (Mané-Padrós, 2010).

In summary, these results led a model for developmentally regulated PCD in the hemimetabolous B. germanica. In this model, JH is responsible to hold back the degeneration of the prothoracic gland during nymphal-nymphal transitions. The low levels of BgFTZ-F1 observed at the end of the penultimate nymphal instar would be below a critical threshold necessary to overcome the BgIAP1-mediated inhibition of prothoracic gland degeneration. During the last nymphal instar, however, the specific and dramatic up-regulation of BgFTZ-F1 in the prothoracic gland at the end of the instar (12 fold higher than in the penultimate instar) would overcome, in the absence of JH, the inhibitory effect of BgIAP1 making the prothoracic gland competent to execute the cell death program (Mané-Padrós, 2010).

This study shows that prothoracic gland degeneration in the nymphal-adult transition of B. germanica show conserved but also divergent features with respect to the highly derived and thoroughly studied species Drosophila melanogaster. This underlines the importance of investigating basal, less modified species, in order to understand the evolutionary trends and mechanisms that led to the highly sophisticated holometabolan mode of metamorphosis (Mané-Padrós, 2010).

Rev-erbα, a heme sensor that coordinates metabolic and circadian pathways

The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. This study shows that heme binds reversibly to the orphan nuclear receptor Rev-erbα, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbα-mediated gene repression. Thus, Rev-erbα serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism (Yin, 2007).

Tests were performed to see whether the heme-dependent recruitment of the NCoR-HDAC3 corepressor complex affected expression of circadian and metabolic Rev-erbα target genes. Consistent with biochemical findings, heme depletion significantly increased the expression of the core clock gene Bmal1, whereas hemin treatment significantly suppressed Bmal1 expression, indicating that intracellular heme concentrations might regulate this Rev-erbα target. Hemin treatment also repressed transcription of the PEPCK and G6Pase genes in human HepG2 liver cells. Conversely, heme depletion by knockdown of ALAS1 significantly induced G6Pase expression and in a manner that was reversed by addition of hemin, demonstrating the dependence of G6Pase transcription on heme concentrations. The repressive effect of heme was abrogated when the abundance of Rev-erbα was reduced by siRNA, indicating that the heme effect was Rev-erbα-dependen. Moreover, heme-dependent recruitment of NCoR and HDAC3, and a concomitant reduction in histone acetylation, was observed by ChIP at the endogenous G6Pase gene. Hemin treatment also repressed the expression of G6Pase and PEPCK in primary mouse hepatocytes and blunted production of glucose, demonstrating the metabolic relevance of heme binding to Rev-erbα (Yin, 2007).

The circadian expression of Rev-erbα is regulated both transcriptionally, by BMAL1-CLOCK and by Rev-erbα itself, as well as posttranslationally, by glycogen synthesis kinase 3β-mediated phosphorylation and stabilization. This study has demonstrated that alteration of heme modulates the interaction between Rev-erbα and the NCoR-HDAC3 corepressor complex. Heme concentrations oscillate in a circadian manner, and heme is also required by proteins that control various metabolic pathways and biological processes, making it a candidate for integrating circadian clock and metabolic systems. Heme negatively affects BMAL1-NPAS2-dependent transcription activation while enhancing Rev-erbα-mediated transcription repression, providing a potential means of maintaining the amplitude of circadian rhythms (Yin, 2007).

Expression of the gene encoding ALAS1, the rate-limiting enzyme in heme biosynthesis, increased in response to peroxisome proliferator activated receptor coactivator-1α, a regulator of mitochondriogenesis that increases flux through the Krebs cycle. This first and rate-limiting enzyme in heme biosynthesis requires succincyl CoA, a Krebs cycle intermediate. Gluconeogenesis competes with the Krebs cycle for metabolic intermediates whose depletion compromises heme biosynthesis as well as mitochondrial oxidative metabolism. The ability of Rev-erbα to function as a receptor for heme could provide a general mechanism for coordinating these processes (Yin, 2007).

PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. A Drosophila model has been developed of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, a drug screening strategy was designed based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, the PPARgamma agonist pioglitazone was found to be neuroprotective in Drosophila. This study shows that pioglitazone can rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS it was found that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, this study showed that the predicted Drosophila PPARγ homologs, E75 and E78 are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, a set of metabolites was identified that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, these data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, these data also identifies several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials (Joardar, 2014).


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