The Drosophila beta FTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone

The acquisition of competence is a key mechanism for refining global signals to distinct spatial and temporal responses. The molecular basis of competence, however, remains poorly understood. Here, we show that 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 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. This study define 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.     

Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein

The E74 early ecdysone-inducible gene plays a key role in the regulatory hierarchy activated by ecdysone at the onset of Drosophila metamorphosis. We show here that E74A protein binds to three adjacent sites in the middle of the E74 gene. The consensus sequence for E74A protein binding, determined by random-sequence oligonucleotide selection, contains an invariant purine-rich core sequence, C/AGGAA. This sequence is also present in the binding sites of two mammalian proteins that, like E74A, are related to the ets oncoprotein. Antibody staining of larval salivary gland polytene chromosomes revealed that E74A protein binds to both early and late ecdysone-inducible puffs. This study supports Ashburner's proposal that the early puffs encode site-specific DNA binding proteins that directly interact with the early and late ecdysone-inducible puffs.     

DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.     

Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also 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.     

Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis

The lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of Caenorhabditis elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila [Nature 408 (2000), 86]. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development [Curr. Biol. 12 (2002), 735]. Here, we ask whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. We find that let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an approximately 500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. We conclude that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone-EcR pathway.     

Influence of the genotype on the polytene chromosome puffing and bioelectrical properties of cellular nuclei of Drosophila melanogaster treated with ecdysone in vivo and in vitro experiments

Activity of polytene chromosome puffs at the 0-hour prepupae stage and bioelectric properties of cellular nuclei of salivary glands were investigated under the influence of the hormone 20-OH-ecdysone in vitro in highly inbred selected lines LA (low activity) and HA (high activity) and their F1 hybrids LA x HA and Ha x LA. The inadaptive line LA differs from the line HA by the smaller size of the puffs. The hybrids exceed the best parental line by the size of the majority of the investigated puffs. In the course of investigation of F1 hybrids Ha x LA chromosomes the phenomenon of heteropuffing has been revealed at the asinapsis sites. In vitro impact of ecdysone increases the electrokinetic potential of cellular nuclei; in hybrids this effect expressed more strongly than in parental lines. The data obtained indicate genetic differences in puffing activity in Drosophila polytene chromosomes as a result of inbreeding, destabilizing selection and heterosis effect, and they also confirm the correlation of bioelectric properties of the nuclear genome with regulation of genetic activity of a cellular nucleus.     

Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.     

Genetic analysis of the Drosophila 63F early puff. Characterization of mutations in E63-1 and maggie, a putative Tom22

The 63F early puff in the larval salivary gland polytene chromosomes contains the divergently transcribed E63-1 and E63-2 ecdysone-inducible genes. E63-1 encodes a member of the EF-hand family of Ca(2+)-binding proteins, while E63-2 has no apparent open reading frame. To understand the functions of the E63 genes, we have determined the temporal and spatial patterns of E63-1 protein expression, as well as undertaken a genetic analysis of the 63F puff. We show that E63-1 is expressed in many embryonic and larval tissues, but the third-instar larval salivary gland is the only tissue where increases in protein levels correlate with increases in ecdysone titer. Furthermore, the subcellular distribution of E63-1 protein changes dynamically in the salivary glands at the onset of metamorphosis. E63-1 and E63-2 null mutations, however, have no effect on development or fertility. We have characterized 40 kb of the 63F region, defined as the interval between Ubi-p and E63-2, and have identified three lethal complementation groups that correspond to the dSc-2, ida, and mge genes. We show that mge mutations lead to first-instar larval lethality and that Mge protein is similar to the Tom22 mitochondrial import proteins of fungi, suggesting that it has a role in mitochondrial function.     

Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster

Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.