Functional analysis of regulatory elements controlling the expression of the ecdysone-regulated Drosophila ng-1 gene.

The steroid hormone ecdysone controls multiple aspects of insect development, including larval moults and metamorphosis, and can induce specific genetic responses in different tissues. The definition of the molecular mechanisms able to mediate this tissue-specific responsiveness may greatly contribute to understanding how such an accurate genetic response is achieved. In this work we have identified, by transgenic analysis, the regulatory elements directing the expression of ng-1, an ecdysone-regulated Drosophila gene showing a highly specific developmental expression profile. Our results show that an ecdysone-responsive element located within the ng-1 coding region is necessary for high-level gene expression, whereas the gene's spatial and temporal expression profile is fully controlled by a distinct upstream regulatory region. This region binds a set of transcriptional factors, including the FKH regulatory protein, which can potentially modulate the ecdysone genetic regulated response.

The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis.

Drosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. In this paper, we focus on the role of ecdysone in regulating adult wing development during metamorphosis. We show that mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion, indicating a role for ecdysone in these morphogenetic responses. We also show that crol and EcR mutations interact with mutations in genes encoding integrin subunits-a family of alphabeta heterodimeric cell surface receptors that mediate cell adhesion in many organisms. alpha-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of alpha- and beta-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. Finally, we describe a new hypomorphic mutation in EcR which indicates that different EcR isoforms can direct the development of adult appendages. This study provides evidence that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. We also propose that ecdysone modulation of integrin expression might be widely used to control multiple aspects of adult development.

crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis.

Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis.

Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes.

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences, the ecdysone response elements (EcREs), whose repertoire still remains to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), we have analysed the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. We report here that the ng-EcRE contacts the ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and that this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and beta FTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive nuclear receptors cross-talking along the development, a mechanism that might play a relevant role in determining the temporal and spatial specificity of the ecdysone response. Finally, we show that the ng-EcRE can promote functional interactions in vitro as well as in vivo, acting as a transcriptional enhancer able to confer a specific developmental expression profile to a minimal promoter in transgenic flies.

A steroid-triggered switch in E74 transcription factor isoforms regulates the timing of secondary-response gene expression.

The steroid hormone 20-hydroxyecdysone (referred to here as ecdysone) directs Drosophila metamorphosis by activating a series of genetic regulatory hierarchies. ETS domain transcription factors encoded by the ecdysone-inducible E74 early gene, E74A and E74B, act at the top of these hierarchies to coordinate the induction of target genes. We have ectopically expressed these E74 isoforms to understand their regulatory functions during the onset of metamorphosis. We show that E74 can regulate its own transcription, most likely through binding sites within its gene. Ectopic expression of E74B can partially repress the E78B and DHR3 orphan receptor genes, suggesting a role for E74 in the appropriate timing of early-late gene expression. Furthermore, E74A is both necessary and sufficient for E78B induction, implicating E74A as a key regulator of E78B expression. We also show, consistent with our studies of E74 loss-of-function mutations, that E74B is a potent repressor of late gene transcription and E74A is sufficient to prematurely induce the L71-1 late gene. However, ectopic expression of both Broad-Complex and E74A activators in an E74B mutant background is not sufficient to prematurely induce all late genes, indicating that other factors contribute to this regulatory circuit. These observations demonstrate that the steroid-triggered switch in E74 transcription factor isoforms plays a central role in the proper timing of secondary-response gene expression.

The moulting hormone ecdysone is able to recognize target elements composed of direct repeats.

In Drosophila melanogaster, three temporally distinct ecdysone-responsive puff sets, the so-called intermoult, early and late puffs, have been described on the salivary gland polytene chromosomes. We have analyzed in detail a DNA segment of the 3C polytene region, from which the originates one of the most prominent intermoult puffs, with the aim of identifying ecdysone response elements (EcREs). Here we report that two putative EcREs of identical sequence are located at this puff site. Interestingly, these elements display a novel structural feature, being composed of directly repeated half-sites. Our results show that the EcR/USP heterodimer known to constitute the ecdysone functional receptor complex is able to bind to and transactivate through target elements composed of directly repeated half-sites. In addition, we show that these elements are also able to bind efficiently USP alone, suggesting that USP and EcR/USP could compete for their binding to DNA.

The role of the BR-C locus on the expression of genes located at the ecdysone-regulated 3C puff of Drosophila melanogaster.

During the third larval instar, the steroid moulting hormone ecdysone activates three temporally distinct puff sets on the D. melanogaster salivary gland polytene chromosome: the so-called intermoult, early and late puffs. Hormonal regulation of intermoult puffs is quite complex and, so far, largely not understood. In order to further investigate this aspect, we have analysed the effects of mutations in a key regulator of the ecdysone response at the onset of metamorphosis, the Broad-Complex (BR-C) locus, on the expression of genes mapping at the 3C intermoult puff. On the basis of an accurate examination of 3C intermoult gene activity in single, carefully staged, third instar larvae of wild-type and BR-C mutant strains, we were able to subdivide these genes into two groups. Each group is characterised by a different temporal expression profile, so that at the beginning of the wandering stage the transcription of the first group declines as group II transcription is induced. Interestingly, the BR-C locus appears to play a regulatory role in establishing this transcriptional switch. By using mutants of each of the three lethal complementation groups, we precisely defined the role of BR-C functions in this developmental transition and we show that this locus also plays an essential role in the early pre-metamorphic hormonal response.

Dense cluster of genes is located at the ecdysone-regulated 3C puff of Drosophila melanogaster.

The 3C11-12 polytene bands of the Drosophila melanogaster X chromosome give rise to a prominent puff, whose regression is triggered by the increase in the titre of the steroid hormone 20-hydroxyecdysone occurring before the metamorphosis. Here, we report the molecular characterization of three genes, named ng-2, ng-3 and ng-4, which we found to be closely linked to each other and to Sgs-4, Pig-1 and ng-1, three other genes previously mapped at this polytene region. All six genes are, in fact, arranged in a tightly linked cluster spanning a DNA segment of only 11 kb. With the exception of ng-4, all the clustered genes are highly expressed only during the larval life and share the same tissue-specificity, being mainly transcribed within the salivary glands. In addition, two members of the cluster, ng-1 and ng-2, show a very high degree of sequence homology, clearly indicating that they are related to each other by means of a duplication event. Interestingly to note, the entire cluster shows a peculiar genomic location, extending across two introns of the memory gene dunce, a large gene of Drosophila whose organization has proved to be remarkably complex.

Effect of ecd1 mutation on the expression of genes mapped at the Drosophila melanogaster 3C11-12 intermoult puff.

The Drosophila melanogaster ecd1 mutation causes a severe temperature-sensitive deficiency in the titre of the steroid hormone ecdysone. This mutation was used to investigate the role of ecdysone in both the transcription of the genes mapped at the 3C11-12 intermoult puff region and the puff formation. Thoroughly synchronized ecd1 larvae were shifted to the non-permissive temperature at various times of the development; after 24 or 48 h, the levels of the transcripts derived from Sgs-4, Pig-1 and ng-1, the three genes located at the 3C11-12 polytene bands, were determined. The results showed that the levels of the transcripts encoded by Pig-1 and ng-1 are unaffected by the drop in the ecdysone titre occurring in non-permissive conditions whereas the amount of Sgs-4 mRNA is greatly reduced. These data clearly indicate that transcription of the three genes mapped within the puff region is affected differently by the hormone. Furthermore, ecd1 larvae cultured at the non-permissive temperature show a prominent puff at the 3C11-12 polytene bands, indicating that ecdysone is not essential for puff induction and that puff size is not simply correlated with high-level Sgs-4 transcription.

Molecular organization of the Drosophila melanogaster Pig-1 gene.

The Pre-intermoult gene-1 (Pig-1) of Drosophila melanogaster maps on the X chromosome, at polytene bands 3C11-12, and is nested within the 79 kb intron of the dunce gene. Pig-1 has so far been characterized only preliminarily and its function is still unknown. We analysed the molecular organization of the gene by cDNA clone isolation and sequencing as well as S1 mapping and primer extension analyses. The results obtained reveal that the gene is colinear with its genomic sequence and define the usage of both 5' and 3' alternative sites for Pig-1 transcription; two continuous open reading frames (ORFs) are fully contained within the Pig-1 transcribed region, although several lines of evidence suggest that only the longer ORF is likely to be translated. We also report that the level of Pig-1 transcript is nearly fourfold reduced in a variant strain carrying a deletion within the Pig-1 upstream sequence, thus identifying a regulatory element required for high level gene expression.