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

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1411-1421 ◽  
Author(s):  
J.C. Fletcher ◽  
C.S. Thummel
Keyword(s):  
Salivary Gland ◽  
Critical Role ◽  
Polytene Chromosomes ◽  
Early Gene ◽  
Secondary Response ◽  
Phenotypic Characterization ◽  
Large Set ◽  
Late Genes ◽  
Larval Salivary Gland ◽  
Maximal Induction

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.

Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 613-627 ◽  
Author(s):  
F. Huet ◽  
C. Ruiz ◽  
G. Richards
Keyword(s):  
Salivary Gland ◽  
Gene Networks ◽  
Larval Instar ◽  
Polytene Chromosomes ◽  
Early Gene ◽  
Rt Pcr ◽  
Response Characteristics ◽  
Larval Salivary Gland

The steroid hormone ecdysone orchestrates insect development by regulating gene networks. In Drosophila the most detailed description of ecdysone action is the sequential activation of early and late puffs in the polytene chromosomes of the late larval salivary gland. A number of these early puffs (2B5, 74EF and 75B) contain complex transcription units (Broad-Complex, E74 and E75 respectively) encoding families of regulatory proteins which are expressed in most if not all tissues. In vitro, transcripts of the different isoforms of these early genes as well as the ecdysone receptor (EcR) present varying dose response characteristics (Karim and Thummel, 1992, EMBO J. 11, 4083–4093). We have developed an in vivo approach using a reverse transcription-polymerase chain reaction assay (RT-PCR) so as to visualise these transcripts in the RNA extracted from a single salivary gland. Using one salivary gland lobe for developmental puff staging and the sister lobe for RT-PCR, we have obtained precise developmental profiles for these transcripts and have extended our study to other tissues and stages where puffing studies were not possible. In the salivary gland we have characterised three distinct ecdysone responses. For the mid and late third larval instar responses our results confirm and extend the conclusions of the in vitro studies concerning the temporal expression of the early gene isoforms. The relatively brief prepupal response contains elements in common with each of the larval responses and all three can be explained by the profiles of the respective ecdysone peaks. Interestingly EcR transcripts respond differently during each response. The analysis of different tissues of the same animal reveals subtle differences in the timing of the ecdysone response and isoform expression and suggests that this may reflect tissue differences in the ecdysone profiles. As these molecules have homologues in vertebrates, our analysis may have general implications for the organisation of hormonal responses in vivo.

Genetic Analysis of the Drosophila 63F Early Puff: Characterization of Mutations in E63-1 and maggie, a Putative Tom22

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 229-244
Author(s):  
Martina Vaskova ◽  
A M Bentley ◽  
Samantha Marshall ◽  
Pamela Reid ◽  
Carl S Thummel ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Genetic Analysis ◽  
Polytene Chromosomes ◽  
Reading Frame ◽  
Protein Levels ◽  
Ef Hand ◽  
Larval Salivary Gland ◽  
Complementation Groups ◽  
Inducible Genes ◽  
Early Puff

Abstract 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 Ca2+-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.

A modification of Heidenhain's iron-haematoxylin technique, as used to stain smears of Drosophila larval polytene chromosomes and neuroblast cells

1971 ◽  
Vol 49 (1) ◽  
pp. 132-133 ◽  
Author(s):  
Albert E. Moorman
Keyword(s):  
Acetic Acid ◽  
Salivary Gland ◽  
Methyl Alcohol ◽  
Polytene Chromosomes ◽  
Larval Salivary Gland

Acetic-acid-fixed smears of Drosophila larval salivary gland chromosomes and of neuroblast cells from the larval ganglion undergoing mitosis are prepared by a modification of Heidenhain's iron-haematoxylin technique, in which absolute methyl alcohol is the solvent of all reagents used in the staining process.

p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes

1988 ◽  
Vol 8 (5) ◽  
pp. 1877-1886
Author(s):  
B M Benton ◽  
S Berrios ◽  
P A Fisher
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Salivary Gland ◽  
Indirect Immunofluorescence ◽  
Polytene Chromosomes ◽  
Nuclear Interior ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Larval Salivary Gland

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

Chromocentre polymorphism in polytene chromosomes of Simulium costatum (Diptera: Simuliidae)

Genome ◽  
1989 ◽  
Vol 32 (4) ◽  
pp. 510-515 ◽  
Author(s):  
C. Brockhouse ◽  
J. A. B. Bass ◽  
N. A. Straus
Keyword(s):  
Salivary Gland ◽  
Polytene Chromosome ◽  
Polytene Chromosomes ◽  
Black Fly ◽  
Weinberg Equilibrium ◽  
Larval Salivary Gland ◽  
Hybrid Introgression ◽  
Hardy Weinberg Equilibrium ◽  
Two Populations

The polytene chromosomes of the black fly species Simulium (Nevermannia) costatum are joined at the centromeres in a strongly heterochromatic chromocentre. Examination of the larval salivary gland chromosomes revealed two populations with a unique polymorphism for attachment to the chromocentre involving all centromeres. All three homologous pairs of chromosomes are polymorphic for centromeres that do not join to the chromocentre. Samples from one of these populations were large enough for thorough study. In this population, the attachment polymorphism is in Hardy-Weinberg equilibrium for two of the centromeres and was in the same frequency for 2 successive years of sampling. The polymorphism could be either primary, retained from an ancestral nonchromocentric state, or secondary, evolving independently or introduced via hybrid introgression. The evolution of chromocentres is discussed in the context of species in the Simulium vernum group.Key words: black fly, polytene chromosome, chromocentre, polymorphism, evolution.

scholarly journals The ecdysone-induced puffing cascade in Drosophila salivary glands: a Broad-Complex early gene regulates intermolt and late gene transcription.

Genetics ◽  
1991 ◽  
Vol 129 (1) ◽  
pp. 169-175
Author(s):  
P S Guay ◽  
G M Guild
Keyword(s):  
Salivary Gland ◽  
Salivary Glands ◽  
Transcriptional Activity ◽  
Early Gene ◽  
Late Gene ◽  
Major Salivary Gland ◽  
Late Genes ◽  
Complex Locus ◽  
Complementation Groups ◽  
Broad Complex

Abstract The steroid hormone 20-OH ecdysone triggers a classic and well-defined program of chromosome puffing that is assumed to reflect changes in transcriptional activity in Drosophila salivary glands. Mutations in each of four Broad-Complex locus (BR-C) complementation groups were analyzed for their effects on the expression of other genes that reside in several major salivary gland puffs. RNA blot analysis showed that the rbp function of the BR-C is required for the transcription of six genes in the 71E late puff and is the first demonstration that an ecdysone-induced early gene controls the transcription of late genes within the puffing cascade. In addition, the rbp function is required for the transcription of four intermolt genes (Sgs-3, Sgs-4, Sgs-5 and 71E gene VII). Mutations in the broad, l(1)2Bc and l(1)2Bd functions of the BR-C had no effect on the expression of the genes examined. We propose that the BR-C functions to control transcription at many other salivary gland loci at the beginning of metamorphosis.

Isolation and characterization of fifteen ecdysone-inducible Drosophila genes reveal unexpected complexities in ecdysone regulation

1993 ◽  
Vol 13 (11) ◽  
pp. 7101-7111
Author(s):  
P Hurban ◽  
C S Thummel
Keyword(s):  
Salivary Gland ◽  
Steroid Hormone ◽  
Polytene Chromosomes ◽  
Feedback Regulation ◽  
Primary Response ◽  
Cdna Libraries ◽  
Isolation And Characterization ◽  
Larval Salivary Gland ◽  
Subtracted Cdna Libraries

Our insights into the regulatory mechanisms by which the steroid hormone ecdysone triggers Drosophila melanogaster metamorphosis have largely depended on puffs in the larval salivary gland polytene chromosomes as a means of identifying genes of interest. Here, we describe an approach that provides access to ecdysone-inducible genes that are expressed in most larval and imaginal tissues, regardless of their ability to form puffs in the polytene chromosomes. Several hundred cDNAs were picked at random from subtracted cDNA libraries and subjected to a rapid and sensitive screen for their ability to detect mRNAs induced by ecdysone in the presence of cycloheximide. Of the 15 genes identified in this manner, 2 correspond to early puffs in the salivary gland polytene chromosomes, at 63F and 75B, confirming that this screen functions at the desired level of sensitivity and is capable of identifying novel primary-response genes. Three of the genes, Eig45-1, Eig58, and Eig87, are expressed coordinately with the salivary gland early genes; one of them, Eig58, maps to the 58BC puff that is active when the 74EF and 75B early puffs are at their maximal size. Another gene identified in this screen, Eig17-1, encodes a novel cytochrome P-450. On the basis of its sequence identity and temporal profile of expression, this gene may play a role in steroid hormone metabolism and thus could provide a mechanism for feedback regulation of ecdysone production. Although all 15 genes have patterns of transcription that are consistent with ecdysone regulation in vivo, 5 genes do not appear to be induced by the late larval ecdysone pulse. This indicates that ecdysone induction in larval organs cultured with cycloheximide is not always indicative of a primary response to the hormone.

scholarly journals p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes.

1988 ◽  
Vol 8 (5) ◽  
pp. 1877-1886 ◽  
Author(s):  
B M Benton ◽  
S Berrios ◽  
P A Fisher
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Salivary Gland ◽  
Indirect Immunofluorescence ◽  
Polytene Chromosomes ◽  
Nuclear Interior ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Larval Salivary Gland

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

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

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1455-1465 ◽  
Author(s):  
J.C. Fletcher ◽  
K.C. Burtis ◽  
D.S. Hogness ◽  
C.S. Thummel
Keyword(s):  
Gene Expression ◽  
Salivary Gland ◽  
Critical Role ◽  
Polytene Chromosomes ◽  
Transcription Unit ◽  
Loss Of Function ◽  
Phenotypic Analysis ◽  
Pupal Development ◽  
Puparium Formation ◽  
Early Puff

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.

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