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![Time course of ectopic E74 induction. (A) Both w and w; P[hs-E74A] animals were synchronized at puparium formation and aged 4 hr. These prepupae were then heat-shocked at 35C for the number of minutes indicated. RNA was extracted from whole animals, and the endogenous and heat-induced E74A and E74B mRNAs were detected by Northern blot hybridization as described (31). RNA was also extracted from non-heat-induced w; P[hs-E74A] 0-hr prepupae (0 hr pp) as a positive control for normal levels of endogenous E74A mRNA, and from non-heat-induced w; P[hs-E74A] 5-hr prepupae (5 hr pp) as a negative control for E74A expression. (B) Both w and w; P[hs-E74B] animals were synchronized at puparium formation and heat-shocked at 35C for the number of minutes indicated. RNA was extracted from whole animals, and the endogenous and heat-induced E74A and E74B mRNAs detected by Northern blot hybridization. RNA was also extracted from non-heat-induced w; P[hs-E74B] 8-hr larvae (8 hr pp) as a positive control for normal levels of endogenous E74B mRNA, and from non-heat-induced w; P[hs-E74B] 1.5-hr prepupae (1.5 hr pp) as a negative control for E74B expression.](profile/Pier-Davino/publication/14104198/figure/fig1/AS:339676016726029@1457996481843/Time-course-of-ectopic-E74-induction-A-Both-w-and-w-Phs-E74A-animals-were.png)
Time course of ectopic E74 induction. (A) Both w and w; P[hs-E74A] animals were synchronized at puparium formation and aged 4 hr. These prepupae were then heat-shocked at 35C for the number of minutes indicated. RNA was extracted from whole animals, and the endogenous and heat-induced E74A and E74B mRNAs were detected by Northern blot hybridization as described (31). RNA was also extracted from non-heat-induced w; P[hs-E74A] 0-hr prepupae (0 hr pp) as a positive control for normal levels of endogenous E74A mRNA, and from non-heat-induced w; P[hs-E74A] 5-hr prepupae (5 hr pp) as a negative control for E74A expression. (B) Both w and w; P[hs-E74B] animals were synchronized at puparium formation and heat-shocked at 35C for the number of minutes indicated. RNA was extracted from whole animals, and the endogenous and heat-induced E74A and E74B mRNAs detected by Northern blot hybridization. RNA was also extracted from non-heat-induced w; P[hs-E74B] 8-hr larvae (8 hr pp) as a positive control for normal levels of endogenous E74B mRNA, and from non-heat-induced w; P[hs-E74B] 1.5-hr prepupae (1.5 hr pp) as a negative control for E74B expression.
Source publication
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 ec...
Contexts in source publication
Context 1
... transformation (36). One transformant line that efficiently expressed either E74A or E74B under the control of the hsp70 promoter was selected for further characterization. In P[hs-E74A] prepupae, a 3.2-kb E74A transcript accumulates in response to a 10-min heat shock at 35C, and maximal levels of mRNA are induced after a 30-min heat treatment (Fig. 1A). Similarly, a 4.4-kb E74B transcript accumulates in P[hs-E74B] prepupae after a 10-min heat shock, and maximal mRNA levels are obtained by 30 min (Fig. 1B). The amount of hs-E74A mRNA is severalfold higher than that normally seen in newly formed prepupae, whereas the amount of hs-E74B mRNA is significantly higher than endogenous E74B ...
Context 2
... characterization. In P[hs-E74A] prepupae, a 3.2-kb E74A transcript accumulates in response to a 10-min heat shock at 35C, and maximal levels of mRNA are induced after a 30-min heat treatment (Fig. 1A). Similarly, a 4.4-kb E74B transcript accumulates in P[hs-E74B] prepupae after a 10-min heat shock, and maximal mRNA levels are obtained by 30 min (Fig. 1B). The amount of hs-E74A mRNA is severalfold higher than that normally seen in newly formed prepupae, whereas the amount of hs-E74B mRNA is significantly higher than endogenous E74B mRNA in late larvae. No hs-E74A or hs-E74B transcripts are detectable in the absence of heat treatment or in w control animals (Fig. ...
Context 3
... levels are obtained by 30 min (Fig. 1B). The amount of hs-E74A mRNA is severalfold higher than that normally seen in newly formed prepupae, whereas the amount of hs-E74B mRNA is significantly higher than endogenous E74B mRNA in late larvae. No hs-E74A or hs-E74B transcripts are detectable in the absence of heat treatment or in w control animals (Fig. ...
Citations
... The E74B is a primary 20E response gene, and its ectopic expression partially repressed the E78B and DHR3 orphan receptor genes, suggesting its role in the appropriate timing of early-late gene expression. 38 Taken together, these results show that E74B cooperates with 20E to participate in insect growth and development. ...
... Taken together, these findings show that in addition to its function as a key primary response gene of 20E, the E74B gene is also a potent repressor of late gene transcription and plays a central role by directly acting on 20E, thus regulating the growth and development process in insects. 38,42 In conclusion, the E74B gene was cloned and reported for the first time. qRT-PCR showed that it is expressed in different tissues and developmental stages in H. armigera. ...
BACKGROUND
The growth and development transition of insects are mainly mediated by ecdysone. As one of the ecdysone‐induced transcription factors, E74 is involved in many physiological processes of insect growth and development. However, E74 and its function in Helicoverpa armigera remains unclear.
RESULTS
In this study, E74B, a subtype of the E74, was identified for the first time in H. armigera. Bioinformatics analysis showed that H. armigera E74B shared the highest homology with E74B in Bombyx mori, which belongs to the E26 transformation‐specific (ETS) superfamily. The expression profile showed that the transcription level of HaE74B increased in the late stages of fourth to sixth instars compared with the early stages; it was also high in the pupa and midgut. Moreover, we investigated the function of HaE74B through RNA interference and 20E rescue experiments. The results showed silencing of E74B affected the molting and growth of larvae, resulting in the death of more than 60% of larvae. In addition, it also seriously affected the metamorphosis of H. armigera, which reduced the pupae rate, the eclosion rate of the pupae, and fecundity. Application of 20E partially restored the defects in the molting, development and pupae rate of H. armigera.
CONCLUSION
Taken together, these results demonstrated that HaE74B plays a critical role in the growth, development, and metamorphosis of H. armigera, which serves as a molecular target and sets out a theoretical foundation for RNAi‐mediated control of this key pest. © 2023 Society of Chemical Industry.
... The E74 encodes a transcription factor ETS (erythroblast transformation specific), which plays a crucial role in mediating the response to 20-hydroxyecdysone (20E) during gene regulation. It exists in two isoforms, E74A and E74B, with distinct patterns of expression: E74A activates 20E-responsive genes, while E74B represses them (Fletcher et al., 1997). Previously the members of the ETS family have been reported to be involved in immune response in a diverse range of insect species including B. mori and D. melanogaster where they directly govern the expression of antimicrobial peptide genes and affect immune responses (Rus et al., 2013;Tanaka et al., 2012). ...
Culex pipiens demonstrates robust circadian rhythms in adult eclosion, flight activity, mating, and development. These rhythmic patterns are believed to be controlled by the endogenous light-entrainable circadian clock that consists of positive and negative regulators working in a transcription-translation feedback loop. Moreover, these mosquitoes undergo seasonal diapause in exposure to the short photoperiod of late summer or early fall. However, the exact genetic and cellular mechanism behind the clock gene-mediated activity pattern, seasonal time measurement, and subsequent diapause initiation still need to be unraveled. To determine the possible linkage between clock genes and downstream processes, here we employed ChIP-sequencing to identify the direct targets of one of the core clock proteins, Cycle (CYC). The nearest genes with peaks mapping to their 1Kb upstream region of the transcription start site were extracted and scanned for consensus E box sequences, resulting in a dataset comprising the target genes possibly regulated by CYC. Based on the highest fold enrichment and functional relevance, we identified genes relating to five gene categories of potential interest, including peptide/receptors, neurotransmission, olfaction, immunity, and reproductive growth. Of these, we validated fourteen genes with ChIP-qPCR and qRT-PCR. These genes showed a significantly high expression in dusk compared to dawn in concert with the activity level of the CYC transcription factor and are thus strong candidates for mediating circadian rhythmicity and possibly regulating seasonal shifts in mosquito reproductive activity.
... During Drosophila development, prior expression of BR proteins is required for the induction of the early primary-response genes and the EcR protein complex by itself is not sufficient for the activation (22). E74B, one of the E74 isoforms has been shown to be involved in repression of downstream genes in the ecdysone regulatory cascade (49). Therefore, our results, which show that E74 is involved in the repression of certain target genes, are congruent with the above-mentioned reports. ...
Significance
Hematophagous Aedes aegypti mosquitoes spread devastating viral diseases. Upon blood feeding, a steroid hormone, 20-hydroxyecdysone (20E), initiates a reproductive program during which thousands of genes are differentially expressed. While 20E-mediated gene activation is well known, repressive action by this hormone remains poorly understood. Using bioinformatics and molecular biological approaches, we have identified the mechanisms of 20E-dependent direct and indirect transcriptional repression by the ecdysone receptor (EcR). While indirect repression involves E74, EcR binds to an ecdysone response element different from those utilized in 20E-mediated gene activation to exert direct repressive action. Moreover, liganded EcR recruits a corepressor Mi2, initiating chromatin compaction. This study advances our understanding of the 20E-EcR repression mechanism and could lead to improved vector control approaches.
... The candidate ERTF of P0, P12 and P1 is E74A, because its expression preceded that of genes from Groups 3, 4 and 5 in vivo and in vitro. E74A has been recognized as an early response gene that activates late genes (Urness & Thummel, 1990Fletcher et al., 1995Fletcher et al., , 1997. In conjunction with these reports, we speculate that CP genes in Groups 3, 4 and 5 are regulated by E74A. ...
We aimed to explain the reason and function of the successive expression of ecdysone-responsive transcription factors (ERTFs) and related cuticular protein (CP) genes during transformation from larva to pupa. The regulation of the expression of CP genes by ERTFs was examined by in vitro wing disc culture and reporter assay using a gene gun transduction system. Two CP genes that showed expression peaks at different stages-BmorCPG12 at W3L and BmorCPH2 at P0 stage-were selected and examined. Reporter constructs conveying putative BHR3, ßFTZ-F1, BHR39, and E74A binding sites of BmorCPG12 and BmorCPH2 showed promoter activity when introduced into wing discs. In the present study, we showed the functioning of the putative BHR3 and E74A binding sites, together with putative ßFTZ-F1 binding sites, on the activation of CP genes, and different ERTF binding sites functioned in one CP gene. From these, we conclude that BHR3, ßFTZ-F1, and E74A that are successively expressed bring about the successive expression of CP genes, resulting in insect metamorphosis. In addition to this, reporter constructs conveying putative BHR39 binding sites of BmorCPG12 and BmorCPH2 showed negative regulation.
... The ecdysone-inducible early gene E74 (Eip74EF) encodes an ETS (erythroblast transformation specific) transcription factor that mediates the 20E response in gene regulation. In mosquitoes, there are two isoforms, E74A and E74B, that act by differentially activating or repressing 20E-responsive genes (22,23). They share a common C-terminal ETS DNA-binding domain and bind to a consensus core motif C / A GGAA, but have unique ...
Significance
In hematophagous female mosquitoes, each reproductive cycle is linked to a separate blood intake, serving as a foundation for the transmission of dangerous human diseases. During each reproductive cycle, female mosquitoes sequentially feed on carbohydrates and protein (blood). Metabolic flux is alternated to support the reproductive cyclicity. We have established that insulin-like peptides (ILPs), critical for regulating metabolism, are genetically controlled by juvenile hormone (JH) and 20-hydroxyecdysone (20E), the key hormones governing the reproduction of female mosquitoes. CRISPR gene-tagging experiments revealed that the JH and 20E pathways coordinate the production of ILPs. This study has uncovered the link between ILPs and JH and 20E pathways in controlling mosquito metabolism during reproduction of the Aedes aegypti mosquito.
... Involvement of EcRE in Drosophila DDC gene promoter (Chen, Reece, O'Keefe, Engstrom, & Hodgetts, 2002) and Aedes Vitellogenin gene (Martin, Wang, & Raikhel, 2001). Target genes of E74A have been identified in Drosophila (L71-1, Fletcher, D'Avino, &Thummel, 1997, L71-6, Urness, & and those of βFTZ-F1 were almost CP genes (EDG84A, Murata et al., 1996) and (Bombyx; bmACP-6.7, Shiomi et al., 2000Wang, Nita, et al., 2009). ...
Background
We classified cuticular protein genes expressed at prepupal stage in wing discs of Bombyx mori into six groups according to their developmental expression and ecdysone responsiveness. Their expression pattern is suggested to be regulated by ecdysone-responsive transcription factors, whose transcripts showed resemblance with those of cuticular protein gene expression.
Result
Group1 and Group2 CP genes showed peak expression at stage W2. Group3 CP genes showed high expression at stage W3E and W3L and were upregulated by 20E addition, showing a peak 12 h after 20E pulse treatment. Group4 CP gene transcripts started expression from stage V5 and peaked at stage W3L. Some genes showed significant increase 4 or 6 h after 20E addition and were induced 6 h and showed a peak 18 h after the 20E pulse treatment. Group5 CP gene transcripts peaked at the same stage W3L. Some Group5 genes showed significant increase 6 h after the 20E addition, while others were not induced by the 20E addition. These different sub-groups showed different expression profiles in the feeding stage. Transcripts of this group genes were induced 12 h and showed a peak 18 h after the 20E pulse treatment. Group6 CP genes peaked at the stage P0, were not induced by the 20E addition, and showed a peak 24 h after the 20E pulse treatment. Group3, 4, 5, and 6 CP genes are suggested to be regulated by BHR4, BR-C, E74A, and βFTZ-F1, respectively. ERTFs showed different responsiveness to 20E concentration. BR-C was most and E74A was least insensitive. The addition of cycloheximide inhibited BR-C, E74A, and βFTZ-F1 expression depending on the length of treatment after ecdysone pulse treatment, which suggests that BHR4 induced BR-C, E74A, and βFTZ-F1.
Conclusion
Expression patterns of CPs were determined by the ecdysone-responsiveness and the related ERTFs expressed in the prepupal stage in B. mori wing discs.
... In contrast, it marks small lipids droplets in the oenocytes of live inhibitor-fed larvae (D). ecdysone information (Fletcher et al., 1997;Ruaud et al., 2010). After molting, βftz-f1 and E74 expression was markedly reduced in CAY10566-fed larvae (Fig. 8). ...
Fatty acid desaturases are metabolic setscrews. To study their systemic impact on growth in Drosophila melanogaster, we inhibited fatty acid desaturases using the inhibitor CAY10566. As expected, the amount of desaturated lipids is reduced in larvae fed with CAY10566. These animals cease feeding soon after hatching, and their growth is strongly attenuated. A starvation program is not launched, but the expression of distinct metabolic genes is activated, possibly to mobilize storage material. Without attaining the normal size, inhibitor-fed larvae molt to the next stage indicating that the steroid hormone ecdysone triggers molting correctly. Nevertheless, after molting, expression of ecdysone-dependent regulators is not induced. While control larvae molt a second time, these larvae fail to do so and die after few days of straying. These effects are similar to those observed in experiments using larvae deficient for the fatty acid desaturase1 gene. Based on these data, we propose that the ratio of saturated to unsaturated fatty acids adjusts a sensor system that directs feeding behavior. We also hypothesize that loss of fatty acid desaturase activity leads to a block of the genetic program of development progression indirectly by switching on a metabolic compensation program.
... The next set of ecdysone-sensitive puffs has been separated into three distinct groups based on how rapidly they are induced by the large ecdysone pulse: early genes, early-late genes, and late genes. Many early genes encode transcription factors, such as the Broad complex (Br-C), E74, and E75 [114][115][116][117][118][119] . The early genes, in turn, activate expression of the late genes and repress their own expression 120 . ...
The past two decades have witnessed incredible progress toward understanding the genetic and cellular mechanisms of organogenesis. Among the organs that have provided key insight into how patterning information is integrated to specify and build functional body parts is the Drosophila salivary gland, a relatively simple epithelial organ specialized for the synthesis and secretion of high levels of protein. Here, we discuss what the past couple of decades of research have revealed about organ specification, development, specialization, and death, and what general principles emerge from these studies. WIREs Dev Biol 2014, 3:281–300. doi: 10.1002/wdev.140
This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation
Early Embryonic Development > Development to the Basic Body Plan
Invertebrate Organogenesis > Flies
... As a measure for ecdysteroid levels, expression of ecdysteroid target genes E74 (isoform A and B) and E75 (isoform B) was detected. E74A and E74B isoforms exhibit a dynamic response to changing levels of ecdysteroids [32]. E74B is induced by intermediate levels of ecdysteroids, but inhibited by higher levels required to induce the E74A isoform. ...
Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.
... Correspondingly, E74A transcript levels fall when ecdysone concentrations start to decline, and E74B mRNA is repressed by rising hormone titers. This behavioral link between the two isoforms is critical for the proper timing of secondary gene responses (Fletcher, D'Avino, & Thummel, 1997;Karim & Thummel, 1991;Urness & Thummel, 1995). ...
Insect metamorphosis is one of the most fascinating biological processes in the animal kingdom. The dramatic transition from an immature juvenile to a reproductive adult is under the control of the steroid hormone ecdysone, also known as the insect molting hormone. During Drosophila development, periodic pulses of ecdysone are released from the prothoracic glands, upon which the hormone is rapidly converted in peripheral tissues to its biologically active form, 20-hydroxyecdysone. Each hormone pulse has a unique profile and causes different developmental events, but we only have a rudimentary understanding of how the timing, amplitude, and duration of a given pulse are controlled. A key component involved in the timing of ecdysone pulses is PTTH, a brain-derived neuropeptide. PTTH stimulates ecdysone production through a Ras/Raf/ERK signaling cascade; however, comparatively little is known about the downstream targets of this pathway. In recent years, it has become apparent that transcriptional regulation plays a critical role in regulating the synthesis of ecdysone, but only one transcription factor has a well-defined link to PTTH. Interestingly, many of the ecdysteroidogenic transcription factors were originally characterized as primary response genes in the ecdysone signaling cascade that elicits the biological responses to the hormone in target tissues. To review these developments, we will first provide an overview of the transcription factors that act in the Drosophila ecdysone regulatory hierarchy. We will then discuss the roles of these transcriptional regulators in controlling ecdysone synthesis. In the last section, we will briefly outline transcription factors that likely have roles in regulating ecdysone synthesis but have not been formally identified as downstream effectors of ecdysone.
