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C S Thummel

Publications and source records attributed to C S Thummel.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Animals↗
PubMed1997

Dueling orphans--interacting nuclear receptors coordinate Drosophila metamorphosis.

At least seven orphan members of the nuclear receptor superfamily are transcriptionally regulated by the steroid hormone ecdysone and expressed during the onset of Drosophila metamorphosis. A recent paper provides functions for two of these receptors, E75B and DHR3, through trans-regulation and heterodimerization. DHR3 appears to function as a switch that defines the transition from a late larva to a prepupa, and E75B functions as a timer that modulates this transition. This study provides a biological function for orphan receptor interactions during development.

Animals↗
PubMed1997

Steroid regulated programmed cell death during Drosophila metamorphosis.

During insect metamorphosis, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) direct the destruction of obsolete larval tissues and their replacement by tissues and structures that form the adult fly. We show here that larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange and their DNA undergoes fragmentation, indicative of apoptosis. Furthermore, the histolysis of these tissues can be inhibited by ectopic expression of the baculovirus anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. In addition, the diap2 anti-cell death gene is repressed in larval salivary glands as rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. Finally, diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes.

Acridine Orange↗
PubMed1997

Coordination of larval and prepupal gene expression by the DHR3 orphan receptor during Drosophila metamorphosis.

The DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. DHR3 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Here we provide evidence that DHR3 directly contributes to both of these regulatory responses. DHR3 protein is bound to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory genes, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Three DHR3 binding sites were identified downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of DHR3 regulation. Ectopic expression of DHR3 revealed that the polytene chromosome binding pattern is of functional significance. DHR3 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. DHR3 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.

Animals↗
PubMed1996

Molecular characterization of the 71E late puff in Drosophila melanogaster reveals a family of novel genes.

Early metamorphic development in Drosophila melanogaster is initiated by pulses of the steroid hormone ecdysone, which are transduced into tissue-specific transcriptional cascades. This process begins with the hormone-dependent activation of a set of transcription factors (early genes) that, in turn, activate set of tissue-specific effector genes (late genes). The 71E cytogenetic region of the salivary gland polytene genome contains several ecdysone-regulated transcription units. Molecular techniques were used to analyze these genes, their transcriptional program and their evolutionary relatedness. We find that this region contains a cluster of ten coordinately regulated late genes (L71 genes) that are organized as five divergently transcribed gene pairs. Maximum parsimony analysis suggests that an ancestral L71 gene duplicated to form the first gene pair which was, in turn, duplicated to form the set of gene pairs. The L71 gene products form a family of small, chemically basic proteins with a conserved backbone of cysteine residues. In addition, the 71E region contains another gene (I71-1) with the regulatory and biochemical characteristics of the salivary gland intermolt glue proteins.

Amino Acid Sequence↗
PubMed1996

Flies on steroids--Drosophila metamorphosis and the mechanisms of steroid hormone action.

Recent studies have provided new insights into the molecular mechanisms by which the steroid hormone ecdysone triggers the larval-to-adult metamorphosis of Drosophila. Ecdysone-induced transcription factors activate large sets of secondary-response genes and provide the competence for subsequent regulatory responses to the hormone. It seems likely that similar hormone-triggered regulatory hierarchies exist in other higher organisms and that Drosophila is providing our first glimpses of the complexities of these gene networks.

Animals↗
PubMed1995

Molecular analysis of a steroid-induced regulatory hierarchy: the Drosophila E74A protein directly regulates L71-6 transcription.

Steroid hormones orchestrate the growth and development of higher organisms by directing spatially and temporally coordinated programs of gene expression. These changes in gene activity can be visualized in Drosophila by virtue of its giant salivary gland polytene chromosomes. A small set of early puffs are induced directly by the steroid hormone ecdysone. The proteins encoded by these puffs appear to induce many late secondary-response puffs as the animal begins to undergo metamorphosis. Here we report that the ETS domain DNA-binding protein encoded by the E74A early gene directly induces L71-6 late gene transcription. We identify four strong E74A binding sites within the 5' region of L71-6 demonstrate that these sites are essential for proper L71-6 induction at puparium formation. These studies provide a direct link between a steroid-induced transcription factor and the activation of a secondary-response promoter, indicating that steroid signals in higher organisms can be transduced and amplified through regulatory hierarchies.

Animals↗
PubMed1995

Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.

Amino Acid Sequence↗
PubMed1995

Ecdysteroid regulation and DNA binding properties of Drosophila nuclear hormone receptor superfamily members.

Pulses of the steroid hormone 20-hydroxyecdysone (20E) trigger the larval-to-adult metamorphosis of Drosophila by reprogramming gene expression throughout the organism. 20E directly induces a small set of early regulatory genes that repress their own expression and induce a large set of late secondary-response genes. We show here that two members of the Drosophila nuclear hormone receptor superfamily, DHR3 and DHR39, are rapidly induced by 20E, in parallel with the early regulatory genes. Both genes also require protein synthesis at high 20E concentrations for their maximal induction by the hormone. Developmental Northern blot analysis reveals that DHR39 is induced in mid third instar larvae and expressed throughout most of third instar larval and prepupal development, while DHR3 is briefly expressed in late third instar larvae and early prepupae. The 20E-induction and temporal patterns of DHR3 and DHR39 transcription strongly suggest that these genes function together with the early regulatory genes to coordinate the complex gene networks that direct the early stages of Drosophila metamorphosis. In an initial effort to understand how these two orphan receptors might function during development, we examined their DNA binding properties and compared them with the known Drosophila nuclear receptor superfamily members that are involved in the ecdysteroid response: EcR, Usp, E75A, E78A, and beta FTZ-F1. Upon testing all pairwise combinations of these seven proteins on a panel of seven oligonucleotides, only EcR and Usp bound DNA as a heterodimer, indicating that this interaction is highly specific. With the exception of E78A, which did not bind any sequence tested, each of the remaining proteins is able to bind to a single consensus AGGTCA half-site; however, each displayed different specificities depending on the flanking nucleotide sequence. These observations suggest that the 20E-regulated orphan receptors function as monomers to control the expression of their target genes.

Animals↗
PubMed1995

The Drosophila E93 gene from the 93F early puff displays stage- and tissue-specific regulation by 20-hydroxyecdysone.

Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis. This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes. Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis. In an effort to address this question, we have isolated and characterized the E93 gene responsible for the stage-specific 93F early puff. The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff. In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation. E93 spans at least 55 kb of genomic DNA and encodes a 146-kDa protein that has no matches in the sequence databases, but displays several characteristics of known Drosophila transcription factors. We propose that E93 acts in a stage-specific regulatory hierarchy in the salivary gland to direct its histolysis in response to the prepupal ecdysteroid pulse.

Amino Acid Sequence↗
PubMed1995

The ecdysone-inducible Broad-complex and E74 early genes interact to regulate target gene transcription and Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone initiate Drosophila metamorphosis by inducing widespread changes in gene expression. The Broad-Complex (BR-C) and E74 are induced directly by ecdysone and encode families of transcription factors that regulate ecdysone primary- and secondary-response genes. Genetic analyses have revealed that mutations in the BR-C and E74 are lethal during metamorphosis and that these mutations cause some similar lethal phenotypes and alterations in secondary-response gene transcription. To examine whether the BR-C and E74 function together during development, we have combined representative alleles from each BR-C and E74 complementation group. Analysis of the morphological and molecular phenotypes of the double-mutant animals reveals that BR-C and E74 alleles act together to produce both novel and synergistic effects. We find that the BR-C and E74 share functions in puparium formation, pupation and early gene induction. In addition, our evidence suggests that the BR-C and E74 transcription factors may directly interact to regulate the expression of salivary gland glue and late genes. This data is consistent with current models which propose that combinations of ecdysone primary-response genes regulate common morphogenetic pathways during insect metamorphosis.

Alleles↗
PubMed1995

The Drosophila E74 gene is required for the proper stage- and tissue-specific transcription of ecdysone-regulated genes at the onset of metamorphosis.

The steroid hormone ecdysone directly induces a small set of early genes, visible as puffs in the larval salivary gland polytene chromosomes, as it signals the onset of Drosophila metamorphorsis. The products of these genes appear to function as regulators that both repress their own expression and induce a large set of secondary-response late genes. We have identified recessive loss-of-function mutations in the early gene E74, a member of the ets protooncogene family that encodes two related DNA-binding proteins, E74A and E74B. These mutations cause defects in pupariation and pupation, and result in lethality during metamorphosis. Here we extend our phenotypic characterization of the E74A and E74B mutant alleles to the molecular level by examining their effects on the transcription of over 30 ecdysone-regulated genes. We show that the transcription of most ecdysone primary-response genes during late larval and prepupal development is unaffected by the E74 mutations. Rather, we find that E74 is necessary for the appropriate regulation of many ecdysone secondary-response genes. E74B is required for the maximal induction of glue genes in mid third instar larval salivary glands, while E74A is required in early prepupae for the proper timing and maximal induction of a subset of late genes. E74 activity is also necessary for the correct regulation of genes expressed predominantly in the fat body, epidermis or imaginal discs. These observations confirm that E74 plays a critical role in regulating transcription during the early stages of Drosophila metamorphosis. In addition, the widespread effects of the E74 mutations on transcription indicate that E74 functions in regulatory hierarchies not only in the larval salivary gland, but throughout the entire organism.

Alleles↗
PubMed1995

The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone.

The steroid hormone ecdysone initiates Drosophila metamorphosis by reprogramming gene expression during late larval and prepupal development. The ecdysone-inducible gene E74, a member of the ets proto-oncogene family, has been proposed to play a key role in this process. E74 is encoded within the 74EF early puff and consists of two overlapping transcription units, E74A and E74B. To assess the function(s) of E74 during metamorphosis, we have isolated and characterized recessive loss-of-function mutations specific to each transcription unit. We find that mutations in E74A and E74B are predominantly lethal during prepupal and pupal development, consistent with a critical role for their gene products in metamorphosis. Phenotypic analysis reveals that E74 function is required for both pupariation and pupation, and for the metamorphosis of both larval and imaginal tissues. E74B mutants are defective in puparium formation and head eversion and die as prepupae or cryptocephalic pupae, while E74A mutants pupariate normally and die either as prepupae or pharate adults. We have also investigated the effects of the E74 mutations on gene expression by examining the puffing pattern of the salivary gland polytene chromosomes in newly formed mutant prepupae. Most puffs are only modestly affected by the E74B mutation, whereas a subset of late puffs are sub-maximally induced in E74A mutant prepupae. These observations are consistent with Ashburner's proposal that early puff proteins induce the formation of late puffs, and define E74A as a regulator of late puff activity. They also demonstrate that E74 plays a wide role in reshaping the insect during metamorphosis, affecting tissues other than the salivary gland in which it was originally identified.

Animals↗
PubMed1995

The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca(2+)-binding protein.

Pulses of ecdysone at the end of Drosophila larval development dramatically reprogram gene expression as they signal the onset of metamorphosis. Ecdysone directly induces several early puffs in the salivary gland polytene chromosomes that, in turn, activate many late puffs. Three early puffs, at 2B5, 74EF, and 75B, have been studied at the molecular level. Each contains a single ecdysone primary-response gene that encodes a family of widely expressed transcription factors. We report here a molecular characterization of the 63F early puff. Unexpectedly, we have found this locus to be significantly different from the previously characterized early puff loci. First, the 63F puff contains a pair of ecdysone-inducible genes that are transcribed in the larval salivary glands: E63-1 and E63-2. Second, E63-1 induction in late third instar larvae appears to be highly tissue-specific, restricted to the salivary gland. Third, E63-1 encodes a novel Ca(2+)-binding protein related to calmodulin. The discovery of an ecdysone-inducible Ca(2+)-binding protein provides a foundation for integrating steroid hormone and calcium second messenger signaling pathways and generates an additional level for potential regulation of the ecdysone response.

Amino Acid Sequence↗
PubMed1994

A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone.

Two successive pulses of ecdysone signal the ends of larval and prepupal development in Drosophila, inducing early and late puffs in the salivary gland polytene chromosomes. Early puff induction in prepupae is dependent on a preceding period of protein synthesis and low ecdysone concentration. We demonstrate here that the competence acquired during this interval can be provided by beta FTZ-F1, a nuclear hormone receptor superfamily member derived from the 75CD mid-prepupal puff. We show that beta FTZ-F1 represses its own transcription and is repressed by ecdysone, explaining its brief expression in mid-prepupae. We further show that ectopic beta FTZ-F1 expression leads to enhanced levels of ecdysone-induced BR-C, E74, and E75 early gene transcription and premature induction of the stage-specific 93F early puff and E93 transcription. These findings indicate that beta FTZ-F1 plays a central role in the prepupal genetic response to ecdysone and provide a molecular mechanism for stage-specific responses to steroid hormones.

Animals↗
PubMed1993

The Drosophila 78C early late puff contains E78, an ecdysone-inducible gene that encodes a novel member of the nuclear hormone receptor superfamily.

We report the molecular definition of an early late puff locus, at position 78C, that is inducible by ecdysone at the onset of Drosophila metamorphosis. This puff contains a single ecdysone-inducible gene consisting of two nested transcription units, E78A and E78B. E78A mRNA is expressed during a brief interval in mid-pupal development and encodes a novel member of the nuclear hormone receptor superfamily. E78B encodes a truncated receptor isoform that lacks the DNA-binding domain and is predominantly expressed at puparium formation and immediately following E78A in pupae. E78B is directly inducible by ecdysone in late third instar larvae and depends on ecdysone-induced protein synthesis for its maximal level of expression. These observations indicate that E78 represents a distinct subset of early ecdysone-inducible regulatory genes.

Amino Acid Sequence↗