Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration

The response of the three major classes of puff in salivary gland chromosomes of larval Drosophila melanogaster to varying β-ecdysone concentrations has been studied in in vitro cultured glands. Two (25AC and 68C) of the intermolt puffs regress at a rate dependent upon the hormone concentration. Three rapidly reacting puffs (23E, 74EF and 75B) respond in a graded way to β-ecdysone concentrations over a range of at least 600 ×. In contrast, five late-reacting puffs (62E, 78D, 22C, 63E, and 82F) do not respond below 5 × 10^?8M and at 2.5 × 10^?7M react maximally. The 50% response of the early puff sites 74EF and 75B and of the late puff sites occurs at 1 × 10^?7M. Two points are discussed in detail: whether ecdysone is necessary as a sustained stimulus or only as a trigger for the sequential puffing response and an evaluation of the absolute ecdysone concentration necessary for induction.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. VI. Inhibition by juvenile hormones.

In the salivary gland chromosomes of late-third instar larvae and in late (8- to 12-hr) prepupae of Drosophila melanogaster, there are ecdysone-induced sequences of puffing patterns which can be reproduced in vitro. These two sequences are separated by a period when the glands are thought to be exposed to a low titer of β-ecdysone and during which they acquire the competence to respond to ecdysone at the late prepupal puff sites. Attempts to modify either the late larval or the late prepupal responses to ecdysone in vitro by the simultaneous addition of juvenile hormone (JH) with ecdysone, to larval or prepupal glands, respectively, are unsuccessful. If, however, JH (ca. 10^?6M) is added to larval glands cultured 6 hr in ecdysone and then 3 hr in JH alone, the subsequent induction of prepupal ecdysone puffs is inhibited. Thus the role of JH appears to lie in modifying the acquisition of competence to respond to ecdysone rather than in a direct antagonism between the two hormones.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster,: IV. The mid prepupal period

In the mid prepupal period of development of Drosophila melanogaster two major changes in gene activity occur in the salivary glands: (a) The mid prepupal puffs (e.g., 63E, 75CD) are induced and (b) the late prepupal puffs (e.g., 62E, 74EF, 75B, and 93F) acquire the competence to respond to ecdysone. These events can be studied in vitro. Both require that the ecdysone titre be very low (<5 × 10^?9, M) and both events depend upon protein synthesis.     

Cytogenetic analysis of the 2B3-4-2B11 Region of the X chromosome of Drosophila melanogaster

Larvae homozygous or hemizygous for the l(l) t435 mutation located within the early ecdysteroid puff 2B5, or carrying a deletion of the 2B5 band, die at the end of the third larval instar. In the salivary gland chromosomes of these larvae only intermoult puffs are detected. If these salivary glands are incubated in vitro with 20-OH ecdysone for 6 h the intermoult puff 68 C remains large, some early puffs (74EF and 75B) are induced to 30–40% of their normal size, other early (63F) and all late puffs (62E, 78D, 82F and 63E) are not induced at all. Puff 2B5 reaches its normal size but does not regress after 6h incubation with 20-OH ecdysone, as it does in normal stocks. The data obtained in this study show the existence of a locus (or loci) in the band (puff) 2B5 which is necessary for the normal response of the salivary gland chromosomes to the hormone 20-OH ecdysone.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.     

Larval saliva in Drosophila melanogaster: production, composition, and relationship to chromosome puffs.

Drosophila melanogaster salivary glands produce a mucoprotein-containing saliva in the third larval instar. At the time of prepupa formation, the protein component of the saliva is more than 30% of the total gland protein. Electrophoresis of reduced and alkylated saliva proteins in acrylamide gels yields four saliva-specific fractions. Two protein fractions contain strongly linked sugar. The molecular weights of the proteins were ascertained in SDS-acrylamide gels. Molecular weights for two sugar-free fractions were found to be 12 × 10³ and 23 × 10³ and, for one fraction containing little sugar, it probably lies below 100 × 10³. The variability of saliva proteins in 67 wild types of D. melanogaster were investigated. With the help of transplantation experiments, it was shown that the salivary glands synthesize saliva autonomously. Saliva proteins could be electrophoretically demonstrated earliest in the salivary glands of 86- to 88-hr-old larvae. After saliva is discharged from the gland lumen at the beginning of prepupa formation, the glands produce another type of saliva during the entire prepupal stage and also secrete it into the gland lumen. The chromosome puffs in section 3C of the X chromosome and in section 68C in the third chromosome show a behavior that is positively correlated with larval saliva synthesis.     

The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro

The larval salivary gland of Drosophila melanogaster synthesises a complex secretion, known as ‘glue’. which is secreted at puparium formation and then cements the puparium to its substrate. This secretion is made during the third larval instar and is stored in the gland cells as large granules. A few hours before puparium formation it is secreted into the gland's lumen by exocytosis. This process is induced by ecdysone and can be studied in vitro. Secretion is initiated about 3.5 hr after exposure of glands to ecdysone and is complete by 8 hr. The effects of varying the ecdysone concentration, of inhibitors of RNA or protein synthesis, and of withdrawing the hormone at various times after initial exposure on the process of secretion have been studied. We conclude that some event(s) occurring during the first 3 hr exposure to ecdysone is necessary to initiate secretion of the glue into the gland lumen. The possible relationship between this event(s) and the ecdysone induced changes in gene activity (puffs) which occur in the salivary glands at the same time is discussed.     

Purification of Drosophila melanogaster ultraspiracle protein and analysis of its A/B region-dependent dimerization behavior in vitro

Two members of the nuclear receptor superfamily, EcR (ecdysteroid receptor protein) and Usp (Ultraspiracle), heterodimerize to form a functional receptor for the steroid hormone 20-hydroxyecdysone and thus enable it to coordinate morphogenetic events during insect metamorphosis. N-terminally His-tagged Usp was overexpressed in E. coli cells as a non-truncated protein and purified to homogeneity in two chromatographic steps. It was demonstrated that the recombinant receptor specifically binds the ecdysone response element of the hsp27 gene promoter (hsp27EcRE). Moreover, a highly synergistically formed heterodimeric complex with the DNA-binding domain of EcR was observed on hsp27EcRE, but not on the native Usp response element from the chorion s15 gene promoter. Recombinant Usp forms homodimers and homotetramers in the absence of DNA, as judged from gel filtration and chemical crosslinking experiments. Truncation of its N-terminal A/B region changes molecular characteristics of Usp, considerably weakening its oligomerization potential under the same experimental conditions. This contrasts with the results obtained previously for the similarly truncated RXR--a vertebrate homolog of Usp.     

The control of prepupal puffing patterns in vitro; Implications for prepupal ecdysone titres in Drosophilia melanogaster

In late third instar larvae and prepupae of Drosophila melanogaster there is a complex change in puffing patterns in the salivary gland chromosomes. There are two peaks of activity in this period. The first, in larvae, is known to be under the control of the moulting hormone ecdysone. The second, in prepupae, is now shown by the in vitro culture of prepupal glands to be under the specific control of β-ecdysone in a manner similar to the first. A new class of puffs, active between these two peaks, whose induction is inhibited by ecdysone in vitro, is described. The behaviour of these puffs, exemplified by 75CD and 63E, suggests a period of very low ecdysone titre in vivo. The developmental significance of the role of ecdysone during prepupal development is discussed.     

Cellular regulation of ecdysone synthesis by the prothoracic glands of Manduca sexta

The cellular mechanism of action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH), was investigated in vitro using prothoracic glands from the tobacco hornworm, Manduca sexta. An involvement of cyclic AMP (cAMP) in PTTH-stimulated ecdysone synthesis was demonstrated as follows: (a) the steroidogenic effect of PTTH on prothoracic glands of day 3 fifth instar larvae and day 0 pupae was mimicked by agents (1-methyl-3-isobutylxanthine, dibutyryl cAMP and forskolin) which act by increasing intracellular levels of cAMP; and (b) PTTH stimulated the formation of cAMP in glands from both stages in a rapid, dose-dependent manner. However, a significant accumulation of cAMP in response to PTTH occurred only in larval prothoracic glands. In pupal glands, effects of the neuropeptide on cAMP synthesis were seen only in the presence of a phosphodiesterase inhibitor. Although cAMP is involved in PTTH action at both stages, it thus appears that the developmental state of the prothoracic glands influences the degree to which cAMP accumulates in response to the neurohormone. In addition to cAMP, it appears from the following that Ca²⁺ plays an essential role in mediating the steroidogenic effects of PTTH: (a) PTTH-stimulated ecdysone synthesis was blocked by omission of Ca²⁺ from the incubation medium; and (b) ecdysone synthesis was stimulated by the calcium ionophore A23187. Agents which act by increasing intracellular levels of cAMP enhanced ecdysone synthesis equally well in both the presence and absence of extracellular calcium. By contrast, cAMP formation stimulated by both PTTH and A23187 was completely dependent upon extracellular Ca²⁺. The results suggest a primary role for Ca²⁺ in mediating PTTH-stimulated synthesis of cAMP, with the cyclic nucleotide in turn stimulating ecdysone synthesis.     

Ecdysone-Related Biomarkers of Toxicity in the Model Organism Chironomus riparius: Stage and Sex-Dependent Variations in Gene Expression Profiles

Despite being considered a model organism in toxicity studies, particularly in assessing the environmental impact of endocrine disrupting compounds (EDCs) and other chemicals, the molecular basis of development is largely unknown in Chironomus riparius. We have characterized the expression patterns of important genes involved in the ecdysone pathway from embryos to pupa, but specially during the different phases of C. riparius fourth larval instar, according to the development of genital and thoracic imaginal discs. Real-Time PCR was used to analyze: EcR and usp, two genes encoding the two dimerizing partners of the functional ecdysone receptor; E74, an early response gene induced by ecdysteroids; vg (vitellogenin), an effector gene; hsp70 and hsc70, two heat-shock genes involved in the correct folding of the ecdysone receptor; and rpL13, as a part of the ribosomal machinery. Our results show for the first time stage and sex-dependent variations in ecdysone-responsive genes, specially during the late larval stage of C. riparius. The induction in the expression of EcR and usp during the VII-VIII phase of the fourth instar is concomitant with a coordinated response in the activity of the other genes analyzed, suggesting the moment where larvae prepare for pupation. This work is particularly relevant given that most of the analyzed genes have been proposed previously in this species as sensitive biomarkers for the toxicological evaluation of aquatic ecosystems. Identifying the natural regulation of these molecular endpoints throughout the Chironomus development will contribute to a more in-depth and accurate evaluation of the disrupting effects of EDCs in ecotoxicological studies.