ecdysoneless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - ecdysoneless
Cytological map position - 62D2
Function - unknown
Keywords - molting, ecdysone biosynthesis
Symbol - ecd
FlyBase ID: FBgn0000543
Genetic map position - 3-[1.5]
Classification - conserved protein of unknown function
Cellular location - cytoplasmic
Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in Drosophila provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless1 (ecd1) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. ecd is shown to encode an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd1 conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd- clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein (Gaziova, 2004).
The insect steroid ecdysone (E), and primarily its active derivative 20-hydroxyecdysone (20E), is responsible for coordination of embryogenesis, larval molting and metamorphosis, the latter involving differentiation of adult structures from precursor imaginal discs. The generic name ecdysone is used to refer to the Drosophila steroid hormone. Blood-circulating ecdysone induces tissue-specific and temporally restricted proliferation, differentiation and programmed cell death. Numerous studies, directed towards understanding how the ubiquitous hormone governs these diverse cellular responses, culminated in detailed dissection of the regulatory cascade downstream of the ecdysone signal (Gaziova, 2004).
The major and best-studied source of ecdysone in insect larvae is the prothoracic gland, which in Drosophila consists of the lateral lobes of the ring gland. After this part of the ring gland degenerates during metamorphosis, adult ovaries contribute to the whole body steroid titer in Drosophila. The main role of ecdysone in adult females is to regulate vitellogenesis. In addition, ecdysone has been implicated in egg chamber maturation during mid-oogenesis. Inactive ecdysone conjugates are maternally deposited to eggs and are mobilized during mid-embryogenesis by the amnioserosa (Gaziova, 2004).
Recently, several Drosophila genes involved in ecdysone biosynthesis have been cloned. One is dare, a homolog of the human adrenodoxin reductase that is necessary for the reduction of mitochondrial cytochrome P450 (Cyp) enzymes (Freeman, 1999). Two other genes, disembodied (dib) and shadow (sad), encode Cyp C22-and C2-hydroxylases, respectively: these two enzymes are responsible for the final two hydroxylation steps of ecdysone synthesis (Chavez, 2000; Warren, 2002). Ecdysone is the final product of the ring gland, which is secreted to the hemolymph and converted to 20E in peripheral tissues. The Cyp C20-hydroxylase responsible for this conversion is encoded by shade (shd) (Petryk, 2003). The dare, dib and sad genes are all expressed in the larval lateral ring gland and in adult ovaries, and their loss-of-function phenotypes can be fully explained as a consequence of ecdysone deficiency. Thus far, only one steroidogenic factor that is not itself an enzyme, without children (woc), has been identified (Wismar, 2000; Warren, 2001). This gene encodes a zinc finger transcription factor that probably activates expression of the cholesterol 7,8-dehydrogenase that executes the first step of ecdysone biosynthesis. Mutations of woc affect a wide range of tissues, suggesting that its transcriptional function is not restricted to regulating expression of the steroidogenic enzyme. No other regulators of the steroidogenic pathway have been identified thus far (Gaziova, 2004 and references therein).
Among steroid-deficient Drosophila mutations, ecdysoneless1 (ecd1) is used to study ecdysone roles in development. The ecd1 mutation is a recessive, temperature-sensitive allele that reduces whole-body ecdysone titers and causes larval arrest at a restrictive temperature, 29°C (Garen, 1977). The effect of ecd1 on ecdysone production is autonomous, because cultured ecd1 mutant ring glands fail to produce ecdysone when upshifted to 29°C (Henrich, 1987; Dai, 1991; Warren, 1996). Ecdysone production is also interrupted in adult ovaries upshifted to the restrictive temperature (Garen, 1977; ; Warren, 1996). After several days at 29°C, oogenesis pauses at the onset of vitellogenesis; this phenotype can be reversed by lowering the temperature (Audit-Lamour, 1981). Transplantation experiments show that this effect of ecd1 is autonomous to the ovary (Garen, 1977; Gaziova, 2004 and references therein).
Developmental events disrupted in ecd1 mutants include fat body protein synthesis (Lepesant, 1978), progression of the eye-forming morphogenetic furrow (Brennan, 1998), salivary gland glue secretion (Biyasheva, 2001) and motor neuron outgrowth (Li, 2001). These defects have been interpreted as consequences of the mutationally induced ecdysone deficiency. However, Redfern and Bownes (1993) caution that a range of anomalies in ecd1 adults result from an autonomous ecd requirement for cell viability and therefore may not be attributable to ecdysone deficiency (Gaziova, 2004).
It is difficult to discern which of the phenotypes result from the ecd1 mutation directly, and which are the consequence of low ecdysone titer, without knowing the primary defect in the ecdysoneless gene, whose molecular identity remained elusive for over 25 years. The ecd locus is shown in this study to encode a protein whose orthologs in several other species, including humans, have not yet been functionally described. The original ecd1 mutation and three non-conditional lethal alleles have been mapped and assessed for their effects. The Ecd protein has been localized to both the steroidogenic and non-steroidogenic tissues, and its cell-autonomous roles in imaginal discs and ovaries have been demonstrated (Gaziova, 2004).
Although the non-conditional ecd- mutants often die during the ecdysis to the second instar, displaying phenotypes that might imply defective ecdysone production, their lethality cannot be a direct consequence of low blood ecdysone for the following reasons. (1) ecd- animals cannot be advanced to the second molt by 20E feeding, despite the fact that similar doses of 20E are sufficient (i) to avert second instar lethality in mutants for the steroidogenic enzyme Dare (Freeman, 1999) and (ii) to induce pupariation in ecd1 larvae at 29°C. (2) Since some of the ecd- animals die during the transition to the second instar, one would expect that their ecdysone titer would be lower from as early as the first instar. However, no reduction of ecdysone content was found in first instar homozygous ecd2 larvae. (3) Although Ecd is abundant in the lateral ring gland during the third instar, no such expression is seen at earlier stages. By contrast, some other steroidogenic genes, such as dib and sad, are strongly expressed in the ring gland beginning at embryogenesis. Finally, development of ecd2 homozygotes can be completely rescued with ubiquitous Ecd expression but not with Ecd targeted by the Feb36-Gal4 driver to the ring gland and to some other organs (Andrews, 2002). Since Ecd presence in the ring gland cannot postpone the death of ecd-null mutants, Ecd must be required prior to the initiation of the second molt in some other tissues. One could be the nervous system, because patched-driven Ecd promotes further development of the mutants (Gaziova, 2004).
A cell-autonomous effect has been demonstrated for the ecd1 allele during differentiation of the thorax sensory bristles (Sliter, 1989). Unexpectedly, induction of ecd-null mitotic clones in the primordia of the adult thorax, the wing imaginal discs, did not produce any defective bristles. This was probably because no ecd- clones occurred in the adult epidermis. Based on the presence of twin ecd+/+ clones in all imaginal discs and in the adult compound eye, it is concluded that the lost ecd- clones were replaced by proliferation of the surrounding ecd+ cells. Many of the defects seen in temperature-upshifted ecd1 mutants have been ascribed to autonomous cell lethality in the imaginal discs (Redfern, 1983). However, small clones of ecd- cells have been detected in imaginal discs upon induction of recombination during early third larval instar, and ecd- clones also survived in the adult ovary. Thus, the loss of ecd is not generally cell lethal although it reduces the ability of the mutant cells to proliferate at the normal rate. Mosaic analyses provide direct evidence for a cell-autonomous, ecdysone-independent function of ecd, which may underlie the previously described defects in adult morphogenesis (Gaziova, 2004).
Clones of ecd- somatic follicle cells cause profound defects, manifest as fusions of adjacent egg chambers and leading to duplications of the nurse cell set, in some cases with two vitellogenic oocytes present at the opposite poles. Similar polarity defects are caused by perturbing the Delta/Notch signaling that specifies the polar follicle cells (PFC), and by perturbing the JAK/STAT pathway through which these cells establish proper separation between egg chambers. It remains to be tested whether the egg chamber fusions in ecd mosaic ovaries might result from a compromised signaling by the PFC. Follicle cells are thought to be the major site of ecdysone production in the ovary. However, it is difficult to imagine that the relatively small ecd- clones could significantly reduce the ecdysone titer in the female. Therefore it is concluded that, as in the case of imaginal discs, the effects of ecd2 on oogenesis are independent of free-circulating ecdysone (Gaziova, 2004).
Germline clones completely lacking ecd function arrest at pre-vitellogenic stages, probably earlier than egg chambers carrying the ovoD1 mutation, thus showing that ecd is autonomously required for oocyte maturation. This result is consistent with the phenotype of ecd1 mutant ovaries: ecd1 females become sterile after a few days at 29°C, with a majority of egg chambers at pre-vitellogenic stages (Audit-Lamour, 1981). Interestingly, the steroidogenic enzyme Dare, and the ecdysone response proteins EcR and E75, are similarly required in the nurse cells for egg maturation, since germline clones mutant for these genes arrest as pre-vitellogenic egg chambers as well. This has led to a proposal that ecdysone synthesis by the germline is necessary in an autocrine manner for the progression of oocytes to the vitellogenic stage. Since normal ecd function is required for autonomous ecdysone production by the ovary (Garen, 1977), the pre-vitellogenic arrest of the ecd- germline clones is consistent with an autocrine germline function (Gaziova, 2004).
By inducing ecd2 mutant clones in adult females, mosaic egg chambers were created in which some nurse cells were null for ecd, whereas others carried the ovoD1 dominant mutation that unconditionally blocks oogenesis. Surprisingly, these mixed-genotype egg chambers continued to mature much beyond the phase of arrest caused by either the ecd2 or ovoD1 mutations acting alone. This suggests a functional rescue among the cells within the egg chamber. Since nurse cells are interconnected by ring canals, it is speculated that the ecd+ ovoD1 cells and their ecd-, ovo+ sisters exchange materials that complement them and consequently permit oocyte development. In the light of the autocrine germline hypothesis, an intriguing possibility is that the product of the ecd+ ovoD1 clones might be ecdysone (Gaziova, 2004).
Although the ecdysoneless gene encodes a protein with highly conserved regions, no data was found that would describe the function of these regions and thus enlighten the mode of Ecd action. The only published report has implicated the human ortholog of Ecd, which compensates for the loss of an unrelated yeast protein GCR2 in transcriptional regulation (Deminoff, 2001). An antibody detects Ecd predominantly in the cytoplasm, and thus does not directly support the possibility that Ecd acts at the level of transcription. Yeast two-hybrid studies have been initiated to address the mechanism of Ecd action by identifying its protein partners. Until the exact function of Ecd is known, interpretations of results obtained with the ecdysone-deficient ecd1 mutants should consider its non-steroidogenic effects (Gaziova, 2004).
The presence of Ecd in the late-third instar ring gland is consistent with the steroid deficiency for which ecd1 was originally identified. The ability to induce puparium formation by feeding the non-pupariating ecd1 larvae at 29°C with 20-hydroxyecdysone (20E) (Garen, 1997; Redfern, 1983; Berreur, 1984), suggested that low steroid levels might be the primary cause of arrest at this stage. To test whether the non-conditional ecd mutants could also be rescued by dietary hormone, homozygous second instar ecd2 and ecdl(3)23 larvae were fed with 20E. The feeding of ecd1 larvae at 29°C served as a positive control: 50 µg/ml and 250 µg/ml 20E doses induced pupariation in 26 out of 100, and in 36 out of 100, ecd1 homozygotes, respectively. By contrast, none of 600 ecd2, or 250 ecdl(3)23, larvae progressed beyond their lethal phase when fed 20E. These results strongly imply that ecdysone deficiency alone does not account for the second instar lethality of these mutants. In support of this view, the whole-body ecdysteroid content was not significantly different between ecd2/ecd2 (0.61±0.13 pg/animal) and ecd+ (0.48±0.08 pg/animal) first instar larvae (Gaziova, 2004).
To address the problem of ecd requirement directly, ecd expression was targeted to the steroidogenic part of the ring gland using transgenic UAS-ecd activated by a Gal4 driver, Feb36. As was expected from the ability of exogenous 20E to rescue pupariation of ecd1 homozygotes at 29°C, Ecd expressed under Feb36 allowed formation of defective puparia in around 25% UAS-ecd, ecd2/ecd1 larvae upshifted to 29°C. The ectopic Ecd presence in the ring gland, evident during the second instar, should therefore restore the impaired hormone synthesis and at least postpone the arrest of ecd-null mutants, if disrupted ecdysone production was the sole cause of their death. However, the Feb36-driven Ecd was insufficient to advance UAS-ecd, ecd2/ecd2 larvae even to the second molt. By contrast, the same UAS-ecd construct expressed under a ubiquitous actin-Gal4 driver allowed ecd2 homozygotes to reach adulthood (Gaziova, 2004).
The failure to rescue non-conditional ecd mutants with Ecd targeted to the ring gland, or by 20E feeding, correlates with the absence of Ecd from the ring gland before the third instar. Taken together, the data show that ecd is autonomously required in other organs before it is needed for ecdysone synthesis. To identify the tissue-specific requirement, Ecd was expressed using several other Gal4 drivers. Ecd driven by the patched (ptc) promoter provided a partial rescue: a single copy of ptc-Gal4 allowed ecd2 homozygotes to molt to the third instar; two copies supported formation of defective but tanned prepupae (Gaziova, 2004).
Northern blot analysis of whole animals showed a single ecd transcript, present throughout development. The mRNA is more abundant towards the end of the final larval instar and during metamorphosis; the strongest expression is observed in mature, egg-laying females. In situ hybridization shows that this increase probably results from strong ecd expression in the ovarian nurse cells. The continuous ecd expression was confirmed at the protein level using a specific antibody that detected Ecd from early embryogenesis to adulthood. Ecd was found in unfertilized eggs, showing maternal deposition of the protein. A Western blot of early larvae homozygous for the ecd2 null allele revealed that low levels of the maternal Ecd protein persists into the first larval instar (Gaziova, 2004).
A steroidogenic role of Ecd presumes its presence in the sites of ecdysone synthesis. Staining of late-third instar larvae reveals Ecd expression in the steroidogenic lateral lobes of the ring gland. However, ring glands of late embryos, and first or second instar larvae, did not show prominent staining. Also, the rest of the body displays only a diffuse signal without a restricted pattern. The ring gland temporal profile was corroborated by using a transgenic ß-galactosidase reporter (ecd-lacZ), which was active only in the medial corpora allata region but not in the lateral steroidogenic gland of second instar larvae. This construct strongly labeled the whole ring gland in late third instar. Except for the medial ring gland, not stained with the antibody, the lacZ reporter probably reflected true ecd expression, because it was driven by an ecd upstream genomic region that is sufficient for the rescue of ecd mutants. Besides the ring gland, specific Ecd expression was found in the nervous system, in the imaginal discs and in developing gonads of third instar larvae. In all cases the Ecd protein predominantly resided in the cytoplasm (Gaziova, 2004).
During metamorphosis the lateral ring gland degenerates. Other organs, such as ovaries, serve as sources of adult ecdysone. In adult ovaries, Ecd protein is expressed in both the somatic follicle cells and the germline nurse cells throughout oogenesis. The signal is stronger in the nurse cells of egg chambers staged 8-10, probably because of the deposition of the Ecd protein, as well as mRNA into the oocyte at this time. High levels of Ecd were detected in the apical part of adult testes, where the somatic and germline stem cells are localized and where spermatogenesis begins. In summary, Ecd expression is detected in the primary steroidogenic organs -- the larval lateral ring gland and the adult ovaries -- as well as in the non-steroidogenic central nervous system and imaginal discs (Gaziova, 2004).
To determine the character of mutations in aberrant ecd alleles, the relevant genomic region from ecd mutants has been sequenced. The temperature sensitive, EMS-induced allele ecd1 contains a substitution of the conserved proline 656 to serine, resulting from a C to T transition. All the other examined alleles: ecd2, and the two previously undescribed alleles ecdg24 and ecdl(3)23, produce truncated Ecd peptides. The ecd2 allele contains a C to T transition that converts Q67 to a stop codon. In the gamma-ray induced ecdg24, a four-base-pair deletion causes a frameshift of four amino acids followed by a stop codon. In ecdl(3)23, the premature termination codon results from a C to T transition at Q650. The extent of the presumed Ecd protein truncations suggests that ecd2, at least, is a null allele. In agreement with the described mutations, a specific antibody raised against a central portion of the Ecd protein detected a wild-type sized band on Western blots from third instar ecd1 larvae (29°C), but not from ecd2, ecdg24 or ecdl(3)23 homozygotes approaching their lethal phases. A truncated Ecd product was found in ecdl(3)23 homozygotes (Gaziova, 2004).
The lethal stage of the ecd mutants was examined to establish whether the structural character of the mutations corresponded to their phenotypic effects. The single proline-to-serine substitution in ecd1 is consistent with indications (Henrich, 1993; Sliter, 1989; Gaxiova, 2004) that the mutant gene product retains a residual function. Although most ecd1 homozygotes complete their second molt at 29°C, the majority of the ecd1/Df(3L)R+R2 hemizygotes, and ecd1/ecd2 and ecd1/ecdl(3)23 heteroallelic mutants, die during the second molt, displaying typical molting defects such as double mouth hooks. Among the non-conditional mutants, ecdg24 homozygotes were the most severely affected, and ecdg24/Df(3L)R+R2 larvae arrested during the first molt with unshed cuticles and double mouth hooks. This early lethality could be in part caused by the dominant Roughened (R) mutation, or by another unknown mutation, on the ecdg24-bearing chromosome, since animals lacking most or all of the Ecd protein in ecd2 homozygous or heteroallelic combinations arrest during the second instar. The new ecdl(3)23 mutation is as severe as ecd2. These results suggest that ecd2, ecdg24 and ecdl(3)23 likewise represent ecd-null alleles that completely prevent development beyond the second instar (Gaziova, 2004).
To examine whether ecd plays a cell-autonomous role during development of the adult, mitotic clones homozygous for the null allele ecd2 were generated using the FLP-FRT system. Mutant clones of a non-essential gene, mbf1, located as ecd on the 3L chromosome arm, served as a control. For both genes, wild-type sister clones and the heterozygous background were recognizable by the presence of ubiquitin-driven GFP and the mini white+ gene markers, placed on the homologous chromosome. When induced early during the first larval instar, large mbf1-/- as well as mbf1+/+ clones appeared in the adult compound eyes. By contrast, only ecd+/+ clones were found with ecd2. The lack of ecd2/ecd2 clones was confirmed by staining of imaginal discs, dissected from late third instar larvae: homozygous mutant clones were only found in mbf1 but not in ecd somatic mosaics. No defects were observed in the adult eyes, legs, wings or thorax derived from the imaginal discs where ecd2/ecd2 clones were induced. Since imaginal disc cells normally proliferate throughout larval life, it is assumed that the ecd-/- cells were replaced by their ecd+ neighbors. The loss of Ecd, however, does not seem to be immediately cell-lethal, because small ecd-/- clones could be seen in eye-antennal imaginal discs when induced at the onset of the third instar (Gaziova, 2004).
Ecd clearly plays a role in oogenesis, since the restrictive temperature prevents development of egg chambers beyond stage 8 in ecd1 flies (Audit-Lamour, 1981). To test whether Ecd is autonomously required in the somatic follicle cells, homozygous ecd2 and control mbf1 clones were induced in adult females. Ovaries with ecd-/- clones displayed defective egg chambers with extranumerary nurse cells, often double the normal 15. Staining with an antibody against Orb, a protein that accumulates in the developing oocyte, confirmed that the aberrant egg chambers resulted from fusions of adjacent cysts, and not by overproliferation of the germline cells. Fasciclin III (FasIII), normally expressed by one pair of specific follicle cells at each pole of each egg chamber, was detected only at the opposite ends of a fusion between two egg chambers. Defective egg chambers that had probably fused from several cysts early in their development showed multiple oocyte precursors, as well as FasIII-positive islands of cells. None of these defects occurred in ovaries containing large mbf1 mutant clones. These results show that ecd is required in the follicle cells for normal oogenesis (Gaziova, 2004).
To test for a direct role of Ecd in oocyte development, ecd2/ecd2 germline clones were induced using the FLP-FRT system with the ovoD1 dominant female sterile marker. When recombination was induced during the first larval instar, control ovoD1 females laid eggs, whereas females carrying the ecd2 mutation over ovoD1 did not. Their ovaries contained clonal egg chambers that did not stain with the anti-Ecd antibody and that had arrested prior to vitellogenesis. When recombination was induced in adult females, some of them laid a few eggs (on average 1 per female) 5-6 days later. Ovaries dissected 3 days after the induction contained mosaic egg chambers, in which some nurse cells lacked the Ecd protein, whereas others strongly stained with anti-Ecd antibody. Interestingly, only these ecd+/ecd- egg chambers progressed to vitellogenic stages, whereas those entirely devoid of Ecd arrested very early, showing degeneration of the nurse cells. Apparently the ecd+, ovoD1 nurse cells and their adjacent ecd-, ovo+ sisters mutually rescued each other, thus allowing further development of the oocyte (Gaziova, 2004).
Late in the third instar larval stage of Drosophila, the titer of the steroid hormone ecdysone increases sharply. This increase is blocked in the temperature-sensitive mutant ecd1 after a temperature shift from 20°C to 29°C. The mutant was used to prepare three samples of late third instar larvae with different titers of ecdysone; the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysone-supplemented food, and also high in a third sample that was kept at 20°C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing two-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at five positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [35S]methionine into the larvae at both early and late third instar stages, in order to label proteins synthesized before and after the increase in ecdysone titer. The results indicate that ecdysone induces two major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies, and other proteins are newly synthesized. Attempts to induce prematurely the synthesis of the new proteins by exposing early third instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can respond to ecdysone (Lepesat, 1978).
By in vitro translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, it was shown that ecdysone greatly increases the amount of translatable messenger RNA for one of the newly synthesized proteins. A clone of DNA complementary to the induced messenger RNA has been isolated from a population of bacteriophage carrying segments of the Drosophila genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA·RNA hybridization, about 50 times more complementary poly(A)-RNA was detected in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the messenger RNA. The ecdysone-induced appearance of a major messenger RNA in late third instar larval fat bodies represents a developmental response to ecdysone that appears to be gene-specific, tissue-specific, and stage-specific, and it has exceptionally favorable features for further molecular studies of the control of gene expression by a steroid hormone (Lepesat, 1978).
Ecdysteroid titer and dopa decarboxylase (aromatic-L-amino-acid carboxy-lyase, EC 220.127.116.11) activity were determined throughout the life cycle of Drosophila. Five peaks in the amount of hormone were observed, which preceeded five dopa decarboxylase peaks by times ranging from 5 to 58 hr. Late in the third instar the hormone and enzyme maxima are nearly coincident. The increase in enzyme activity observed at this time is paralleled by an increase in translatable dopa decarboxylase mRNA. To obtain evidence that ecdysterone induces the appearance of this mRNA use was made of the temperature-sensitive ecd1 mutant. When third instar mutant larvae are kept at 29oC, the ecdysteroid titer remains low. In such larvae the normal increase in dopa decarboxylase activity fails to appear, and no translatable dopa decarboxylase mRNA can be detected. Exogenous feeding of ecdysterone to these larvae results in a rapid synthesis of dopa decarboxylase in the epidermal cells. In addition, a parallel increase in translatable dopa decarboxylase mRNA occurs, which may be a primary response of these target cells to ecdysterone (Kraminsky, 1980).
The temperature-sensitive 1(3)ecd-1ts mutation has been used to obtain Drosophila larvae deprived of moulting hormone. The development of mutants and controls during the third larval instar at permissive (20°C) and restrictive temperatures (29°C) was compared. Pupariation is inhibited when larvae are shifted to the restrictive temperature immediately at the second moult. The permanent larvae obtained remain active, do not leave the food, and reach a maximum weight superior to the weight of controls. Ecdysteroids were studied during the third larval instar by HPLC analysis and radioimmunoassays. A careful synchronization of the larvae at the second moult enabled the confirmation that at least one ecdysteroid peak occurs during the third larval instar, prior to the wandering stage in controls (20 or 29°>C). Ecdysone is then the predominant moulting hormone, whereas 20-hydroxyecdysone is the main ecdysteroid at the time of pupariation. Low levels of ecdysteroid were measured in mutant larvae shifted to 29°C immediately at the second moult but larvae completely deprived of immunoreactive material were never observed. Nearly normal levels of ecdysteroids appeared at 27.5°C. Feeding ecd-1 larvae maintained at restrictive temperature on 20-hydroxyecdysone-yeast mixture for 16 hr triggered abortive pupariation. Ecdysteroid levels were measured after the return of the larvae to the standard medium; normal levels were restored 24 hr later. The mutant ecd-1 appears to present interesting opportunities for the detailed study of the hormonal induction of a developmental process during the third larval instar (Berreur, 1984).
Peaks in hsp 26, 28, and 83 RNA levels are correlated with peaks in ecdysteroid titers during mid-embryogenesis, pupariation, and mid-pupation, and with a peak in the level of RNA from the 74EF ecdysone puff at pupariation. Inhibition of the ecdysteroid peak at pupariation by temperature shift of the conditionally ecdysteroid-deficient strain ecd-1 was followed by a disappearance of hsp 26 RNA and a decline in hsp 83 RNA level; subsequent addition of exogeneous 20-OH-ecdysone to the temperature-shifted strain resulted in a severalfold increase in hsp 83 RNA level, and a dramatic increase in that of hsp 26. These results are consistent with the induction of the hsp 83, 28, and 26 genes by ecdysteroid at several developmental stages (Thomas, 1986).
Ring glands dissected from homozygous l(3)ecd1ts wandering larvae and upshifted in vitro to the restrictive temperature, 29°C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29°C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29°C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29°C autonomously and may also interfere with PTTH release (Henrich, 1987).
The temporal patterns of glucose dehydrogenase (Gld) and alcohol dehydrogenase (Adh) expression in Drosophila are correlated positively and negatively, respectively, with ecdysterone titers during the late third instar. In mutant l(3)ecdysone-1ts (ecd-1) larvae, the normal peak of Gld mRNA late in the third instar is not expressed. Conversely, the normal decrease in Adh mRNA at this stage fails to occur in ecd-1. These two abnormal patterns can be reversed by treatment with exogenous ecdysterone. Premature exposure of wild type mid-third instar larvae to ecdysterone also results in the rapid accumulation of Gld mRNA and signals the repression of Adh mRNA. The observed decrease in Adh mRNA expression is accompanied by a transient switch in promoter usage from proximal to distal transcription start sites, which normally occurs later in the third instar (Murtha, 1989).
The temperature-sensitive mutation 1(3)ecd1 of Drosophila is known to autonomously impair the ability of the larval prothoracic gland to produce the steroid molting hormone ecdysone in response to stimulation by the tropic neuropeptide prothoracicotropic hormone. It is shown that autonomous expression of the 1(3)ecd1 mutation in metamorphosing imaginal tissues disrupts the spatial pattern of sensory bristles. Transfer of homozygous mutant animals to the restrictive temperature at the time of pupariation results in the elimination of sensory microchaetae and macrochaetae. This effect is specific to the sensory bristles; nonsensory bristles are not eliminated, nor are other types of innervated cuticular sense organs. In the case of the dorsal thoracic macrochaetae, normal ecd gene function is required during an early period of bristle development (0-18 h after puparium formation at 20°C). It is during this period that important determinative events take place in developing imaginal tissues that are responsible for the establishment of bristle progenitor cells. It is proposed that the ecd gene product may be required for the response of certain classes of cells to specific, regulatory signals (Sliter, 1989).
The ecdysoneless locus in Drosophila has been defined previously by a single conditional mutation, l(3)ecd1, that causes an ecdysteroid deficit and larval death at the restrictive temperature, 29°C, although the primary role of the mutation in developmental processes has been unclear. Gene dosage and complementation studies reported here for ecd1 and five nonconditional lethal alleles indicate that the ecd locus plays prezygotic and postzygotic roles essential for normal embryonic development, the successful completion of each larval molt, adult eclosion, and female fertility. The ecd locus is also required for normal macrochaete differentiation. For each observed phenotype, the severity of mutational effects was correlated with ecd mutant genotypes. In all cases, ecd1 homozygotes were least affected. Mutants heteroallelic for ecd1 and any one of four nonconditional recessive mutations were more severely affected than ecd1 homozygotes, revealing these as hypomorphic alleles. For all phenotypic effects, mutants heteroallelic for ecd1 and a dominant mutation (ecd3D) were most severely affected. These individuals died during embryogenesis at 29°C and developed no macrochaetes on the dorsal thorax when transferred to 29°C during the white prepupal stage. The ecd3D mutation also caused female semisterility in heterozygotes. Ecdysteroid regulation has been implicated previously in all the developmental processes disrupted by these ecd mutations except for macrochaete differentiation (Henrich, 1993).
Studies in vitro have revealed that intact ring glands of Drosophila convert tritiated cholesterol (C) and 25-hydroxycholesterol (25C) via 7-dehydrocholesterol (7dC) and 7-dehydro-25-hydroxycholesterol (7d25C), respectively, to ecdysone (E) and 2-deoxyecdysone (2dE), while both intact and homogenized ovaries synthesize only 2dE from these precursors. Emulsified 7d25C is incorporated directly into ecdysteroids by these tissue preparations at a much greater rate than is 7d25C made in situ from 25C. To probe the basis of the biochemical defect in the ecdysteroid deficient conditional mutant ecdysoneless (ecd1), the differential incorporation into ecdysteroids of C (via 7dC), and particularly of 25C (via 7d25C), was measured relative to that observed after the incubation of 7d25C directly with both wild type and mutant tissues in vitro at 30°C, the restrictive temperature. Both C and 25C were equally 7,8-dehydrogenated in situ to 7dC or 7d25C, respectively, by both wild type and mutant tissues at 30°C. However, the rate of subsequent conversion of either of these delta 5,7-sterol intermediates synthesized in situ to ecdysteroids was reduced an average of 50% in the mutant tissues relative to the wild type. Yet, when emulsified 7d25C was incubated directly with either the wild type or mutant tissues at the restrictive temperature, the amplified rate of conversion of the freely available 7d25C to ecdysteroid by these tissues was identical. These data suggest that the defect in ecd1 tissue-mediated ecdysteroidogenesis does not involve a 'hit' on any of the enzymes involved in either the 7,8-dehydrogenation of C or 25C or in the subsequent oxidation of 7d25C or 7dC to ecdysteroid. Rather, the mutation appears to affect the expression of a gene governing the translocation of delta 5,7-sterol intermediates from the subcellular compartment where they are synthesized and/or stored to the site of subsequent oxidation to ecdysteroid (Warren, 1996).
In Drosophila, secretion of the steroid hormone ecdysone from the prothoracic ring gland coordinates and triggers events such as molting and metamorphosis. In the developing Drosophila compound eye, pattern formation and cell-type specification initiate at a moving boundary known as the morphogenetic furrow. The role of ecdysone in eye development was investigated; the ecdysone signaling pathway is required for progression of the morphogenetic furrow in the eye imaginal disc of Drosophila. Genetic disruption both of the ecdysone signal in vivo with the ecdysoneless1 (ecd1) mutant and of ecdysone response with a Broad-Complex mutant result in disruption of morphogenetic furrow progression. In addition, ecdysone-dependent gene expression, both of a reporter of transcriptional activity of the Ecdysone Receptor and of the Z1 isoform of the Broad Complex, are localized in and close to the furrow. These results suggest that, in the morphogenetic furrow, temporal hormonal signals are integrated into genetic pathways specifying spatial pattern (Brennan, 1998).
Normally the cells mitotic division cycle becomes synchronized in the furrow. Ahead of the furrow, cells proliferate randomly but, in the furrow, all cells are held in G1 arrest. This is followed by a tight band of DNA synthesis and mitosis in all those cells not included in preclusters in the furrow. Attempts were made to determine whether the ecd-ts disruption of the furrow includes this cell cycle regulation. This was tested by two methods and both showed that, while the prefurrow general proliferation is not visibly altered, cell-cycle synchrony in the furrow is lost (Brennan, 1998).
The first method was to use bromodeoxyuridine (BrdU) incorporation to label S-phase cells. A general loss of BrdU incorporation was observed in ecd-ts eye discs, in particular in the zone just posterior to the furrow. The second method was to stain for Cyclin B (CycB) expression, which labels cells at the G2/M transition. In wild-type, CycB is expressed generally anterior to the furrow and in a tight band just posterior to it. In ecd-ts, the anterior CycB expression remains, but the tight, postfurrow band of CycB is lost. Thus both approaches show results consistent with a loss of cell cycle regulation in the furrow of ecd-ts discs. This supports the hypothesis that the eye disc phenotypes seen after loss of ecdysone are not due to general failure of disc cell proliferation, but rather to specific effects on the furrow. In addition, the pattern of cell death in the disc was examined using acridine orange staining; no excess cell death is associated with ecdysone withdrawal. This result differs from previous work (Redfern, 1983) which reported cell death in imaginal discs of ecd1 homozygotes that had been exposed to 29°C for 2 days. It may be that generalized cell death is an effect seen in this genotype only after longer periods of exposure to 29°C (Brennan, 1998).
The results thus far have shown that the morphogenetic furrow requires ecdysone during the middle phase of progression across the eye disc. Mechanisms controlling the earliest phases of furrow initiation and progression are believed to be different from those controlling later phases, so whether the sensitivity of the furrow to ecdysone titer is restricted to the later phases was investigated. By staining early 3rd instar ecd-ts eye discs with 22C10, it was found that even the earliest phases of furrow progression are sensitive to ecdysone. Overly mature photoreceptor clusters with well-extended axonal projections can be found at the anterior edge of differentiation, just as in older discs (Brennan, 1998).
Progression of the furrow beyond the first ten columns is driven by Hedgehog, expressed in the differentiating clusters and diffusing forward to induce anterior cells to enter the furrow. Two eye-specific hedgehog alleles (hh1 and hhfse) cause the furrow to arrest about a third of the way across the eye field, and when Hedgehog is removed with a temperature-sensitive allele, only the first several rows of photoreceptor clusters are formed, yielding a 'Bar' shaped eye. Whether the furrow failure in ecd-ts discs might be associated with loss of Hedgehog expression was examined (Brennan, 1998).
Disc pairs were separated after fixation and stained separately with anti-Ato and anti-Hh antibodies. In all larvae where Ato expression was impaired in the typical ecd-ts manner, Hh protein in the contralateral disc was greatly reduced far below normal levels. Hh protein appears to be lost uniformly across the disc, contrasting with Ato whose domain of expression is progressively narrowed from the anterior (Brennan, 1998).
In the late third larval instar of Drosophila, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29°C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18°C to 29°C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29°C following dissection from wandering third instar larvae reared at 18°C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis (Dai, 1991).
Hormonal regulation in development and maintenance of synaptic transmission involves examination of both the presynaptic and postsynaptic components and a system in which the hormones can be controlled. The ecdysoneless heat-sensitive mutation (l(3)ecd1/l(3)ecd1) of Drosophila was used to provide the ability to regulate endogenous ecdysone production at various larval stages. In conjunction, the neuromuscular junctions of Drosophila were used since they offer the advantage of assessable preparations for both morphological and physiological measures. The growth in the Ib and Is motor nerve terminals and the corresponding muscle 6 in segment 4 of the larval Drosophila throughout the third instar stage in the presence of normal and a much reduced endogenous ecdysone level was investigated. Muscle 6 and the motor nerve terminals parallel in growth throughout the third instar. The nerve terminals increase in length and varicosity number, thus providing an increase in the number of synaptic release sites. The ecdysoneless larvae also show an increase in muscle size, however the Is and Ib motor nerve terminals do not mature to the extent of the wild-type ecdysone producing flies. The motor nerve terminal length is shorter with fewer numbers of varicosities per terminal. In spite of a shorter nerve terminal and fewer varicosities, with an increasing muscle fiber, the compound excitatory junctional potentials of Ib and Is in the ecdysoneless flies are larger, which is suggestive of synaptic structural modification (Li, 2001).
Developmental differences of the motor nerve terminals in the ecdysoneless flies were expected, given that it is known that exogenously applied ecdysone can alter the growth of the mushroom bodies and axon outgrowth in crickets and in Drosophila. It was also noted that the ecdysoneless flies, as used in this study, form enlarged type III synaptic varicosities. It would be of interest to know if the lengths and numbers of varicosities of those terminals are also altered. Some effects of ecdysone appear to be neuronal-specific in their genomic response since growth is promoted in particular neurons, while other neurons retract their process or die with exposure, as was demonstrated for insect neurons in culture. The mechanism of ecdysones action to either increase or decrease neuronal growth is likely through multiple selective genomic actions, since a variety of cellular processes are needed to mediate such actions over a period of days. Because there are diverse actions of ecdysone in vivo and in vitro concerning neuronal development, from promoting growth to death, there may indeed be various isoforms of the ecdysone receptor (EcR) expressed in different neurons, which results in the varied responses, or possibly the EcRs are the same, but translational differences occur. These issues have not yet been resolved in the selective actions of ecdysone on the motor neurons in Drosophila. Although it is conceivable that at different times in insect development when titers of ecdysone may vary substantially, there could be selective actions due to receptor levels and various physiological states of the neurons, as well as competitive actions of other hormones, such as juvenile hormone, which also varies in titer throughout development. Juvenile hormone levels have not been measured in the ecd1/ecd1 larvae when ecdysone titers are low; the ecd mutation may result in altered juvenile hormone production. It is known that different EcR isoforms can be induced by 20-HE during development in Bombyx mori and that the expression of the EcR does vary in the nervous system of Manduca sexta throughout development (Li, 2001 and references therein).
The mechanisms by which an organism becomes immune competent during its development are largely unknown. When infected by eggs of parasitic wasps, Drosophila larvae mount a complex cellular immune reaction in which specialized host blood cells, lamellocytes and crystal cells, are activated and recruited to build a capsule around the parasite egg to block its development. Parasitization by the wasp Leptopilina boulardi leads to a dramatic increase in the number of both lamellocytes and crystal cells in the Drosophila larval lymph gland. Furthermore, a limited burst of mitosis follows shortly after infection, suggesting that both cell division and differentiation of lymph gland hemocytes are required for encapsulation. These changes, observed in the lymph glands of third-instar, but never of second-instar hosts, are almost always accompanied by dispersal of the anterior lobes themselves. To confirm a link between host development and immune competence, mutant hosts, in which development is blocked during larval or late larval stages, were infected. In genetic backgrounds where ecdysone levels are low (ecdysoneless) or ecdysone signaling is blocked (nonpupariating allele of the transcription factor broad), the encapsulation response is severely compromised. In the third-instar ecdysoneless hosts, postinfection mitotic amplification in the lymph glands is absent and there is a reduction in crystal cell maturation and postinfection circulating lamellocyte concentration. These results suggest that an ecdysone-activated pathway potentiates precursors of effector cell types to respond to parasitization by proliferation and differentiation. It is proposed that, by affecting a specific pool of hematopoietic precursors, this pathway thus confers immune capacity to third-instar larvae (Sorrentino, 2002).
Ance is a single domain homologue of mammalian angiotensin-converting enzyme (ACE) and is important for normal development and reproduction in Drosophila melanogaster. Mammalian ACE is responsible for the synthesis of angiotensin II and the inactivation of bradykinin and N-acetyl-Ser-Asp-Lys-Pro, but the absence of similar peptide hormones in insects suggests novel functions for Ance. Evidence is provided in support of a role for Ance during Drosophila metamorphosis. The transition of larva to pupa was accompanied by a 3-fold increase in ACE-like activity, which subsequently dropped to larval levels on adult eclosion. This increase was attributed to the induction of Ance expression during the wandering phase of the last larval instar in the imaginal cells (imaginal discs, abdominal histoblasts, gut imaginal cells and imaginal salivary gland). Ance expression was particularly strong in the presumptive adult midgut formed as a result of massive proliferation of the imaginal midgut cells soon after pupariation. No Ance transcripts were detected in the midgut of the fully differentiated adult intestine. Ance protein and mRNA were not detected in imaginal discs from wandering larvae of flies homozygous for the ecd1 allele, a temperature-sensitive ecdysone-less mutant, suggesting that Ance expression is ecdysteroid-dependent. Physiological levels of 20-hydroxyecdysone induce the synthesis of ACE-like activity and Ance protein by a wing disc cell line confirming that Ance is an ecdysteroid-responsive gene. It is proposed that the expression of Ance in imaginal cells is co-ordinated by exposure to ecdysteroid (moulting hormone) during the last larval instar molt to increase levels of ACE-like activity during metamorphosis. The enzyme activity may be required for the processing of a developmental peptide hormone or may function in concert with other peptidases to provide amino acids for the synthesis of adult proteins (Siviter, 2002).
Human metastatic lymph node 64 (MLN64) is a transmembrane protein that shares homology with the cholesterol-binding vertebrate steroid acute regulatory protein (StAR)-related lipid transfer domain (START) and is involved in cholesterol traffic and steroid synthesis. A Drosophila gene (Start1) has been identified whose putative protein product shows extensive homology with MLN64. The putative Start1 protein, derived from Start1 cDNA sequences, contains an additional 122 aa of unknown function within the StAR-related lipid transfer domain. Similar inserts seem to exist in the Start1 homologues of Drosophila pseudoobscura and Anopheles gambiae, but not in the homologous protein of the urochordate Ciona intestinalis. Immunostaining using an insert-specific antibody confirms the presence of the insert in the cytoplasm. Whereas RT-PCR data indicate that Start1 is expressed ubiquitously, RNA in situ hybridizations demonstrate its overexpression in prothoracic gland cells, where ecdysteroids are synthesized from cholesterol. Transcripts of Start1 are detectable in embryonic ring gland progenitor cells and are abundant in prothoracic glands of larvae showing wave-like expression during larval stages. In adults, Start1 is expressed in nurse cells of the ovary. These observations are consistent with the assumption that Start1 plays a key role in the regulation of ecdysteroid synthesis. Vice versa, the expression of Start1 itself seems to depend on ecdysone, as in the ecdysone-deficient mutant ecd-1, Start1 expression is severely reduced (Roth, 2004).
Biochemical mechanisms that control the levels and function of key tumor suppressor proteins are of great interest as their alterations can lead to oncogenic transformation. This study identified the human orthologue of Drosophila Ecdysoneless (hEcd) as a novel p53-interacting protein. Overexpression of hEcd increases the levels of p53 and enhances p53 target gene transcription whereas hEcd knockdown has the opposite effects on p53 levels and target gene expression. Furthermore, hEcd interacts with Mdm2 and stabilizes p53 by inhibiting Mdm2-mediated degradation of p53. Thus, hEcd protein represents a novel regulator of p53 stability and function. These studies also represent the first demonstration of a biochemical function for hEcd protein and raise the possibility that altered hEcd levels and/or function may contribute to oncogenesis (Zhang, 2006).
Search PubMed for articles about Drosophila ecdysoneless
Audit-Lamour, C. and Busson, D. (1981). Oogenesis defects in the ecd-1 mutant of Drosophila melanogaster, deficient in ecdysteroid at high temperature. J. Insect Physiol. 27: 829-837.
Berreur, P., Porcheron, P., Moriniere, M., Berreur-Bonnenfant, J., Belinski-Deutsch, S., Busson, D. and Lamour-Audit, C. (1984). Ecdysteroids during the third larval instar in l(3)ecd-1ts, a temperature-sensitive mutant of Drosophila melanogaster. Gen. Comp Endocrinol. 54: 76-84. 6427061
Biyasheva, A., Do, T. V., Lu, Y., Vaskova, M. and Andres, A. J. (2001). Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis. Dev. Biol. 231, 234-251. 11180965
Brennan, C. A., Ashburner, M. and Moses, K. (1998). Ecdysone pathway is required for furrow progression in the developing Drosophila eye. Development 125: 2653-2664. 9636080
Chavez, V. M., Marques, G., Delbecque, J. P., Kobayashi, K., Hollingsworth, M., Burr, J., Natzle, J. E. and O'Connor, M. B. (2000). The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development 127: 4115-4126. 10976044
Dai, J. D., Henrich, V. C. and Gilbert, L. I. (1991). An ultrastructural analysis of the ecdysoneless (l(3)ecd1ts) ring gland during the third larval instar of Drosophila melanogaster. Cell Tissue Res. 265: 435-445. 1786592
Deminoff, S. J. and Santangelo, G. M. (2001). Rap1p requires Gcr1p and Gcr2p homodimers to activate ribosomal protein and glycolytic genes, respectively. Genetics 158: 133-143. 11333224
Freeman, M. R., Dobritsa, A., Gaines, P., Segraves, W. A. and Carlson, J. R. (1999). The dare gene: steroid hormone production, olfactory behavior, and neural degeneration in Drosophila. Development 126: 4591-4602. 10498693
Garen, A., Kauvar, L. and Lepesant, J.-A. (1977). Roles of ecdysone in Drosophila development. Proc. Natl. Acad. Sci. 74: 5099-5103
Gaziova, I., Bonnette, P. C., Henrich, V. C. and Jindra, M. (2004). Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis. Development 131: 2715-2725. 15128659
Henrich, V. C., Tucker, R. L., Maroni, G. and Gilbert, L. I. (1987). The ecdysoneless (ecd1ts) mutation disrupts ecdysteroid synthesis autonomously in the ring gland of Drosophila melanogaster. Dev. Biol. 120: 50-55. 3102296
Henrich, V. C., Livingston, L. and Gilbert, L. I. (1993). Developmental requirements for the ecdysoneless (ecd) locus in Drosophila melanogaster. Dev. Genet. 14: 369-377. 8293578
Kraminsky, G. P., et al. (1980). Induction of translatable mRNA for dopa decarboxylase in Drosophila: an early response to ecdysterone. Proc. Natl. Acad. Sci. 77(7): 4175-9. 6776524
Lepesant, J. A., Kejzlarova-Lepesant, J. and Garen, A. (1978). Ecdysone-inducible functions of larval fat bodies in Drosophila. Proc. Natl. Acad. Sci. 75: 5570-5574. 103097
Li, H. and Cooper, R. L. (2001). Effects of the ecdysoneless mutant on synaptic efficacy and structure at the neuromuscular junction in Drosophila larvae during normal and prolonged development. Neuroscience 106: 193-200. 11152710
Murtha, M. T. and Cavener, D. R. (1989). Ecdysteroid regulation of glucose dehydrogenase and alcohol dehydrogenase gene expression in Drosophila melanogaster. Dev. Biol. 135(1): 66-73. 2504635
Petryk, A., Warren, J. T., Marques, G., Jarcho, M. P., Gilbert, L. I., Kahler, J., Parvy, J. P., Li, Y., Dauphin-Villemant, C. and O'Connor, M. B. (2003). Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. Proc. Natl. Acad. Sci. 100: 13773-13778. 14610274
Redfern, C. P. F. and Bownes, M. (1983). Pleiotropic effects of the 'ecdysoneless-1' mutation of Drosophila melanogaster. Mol. Gen. Genet. 189: 432-440
Roth, G.E., et al. (2004). The Drosophila gene Start1: a putative cholesterol transporter and key regulator of ecdysteroid synthesis. Proc. Natl. Acad. Sci. 101(6): 1601-6. 14745013
Sato, T., Jigami, Y., Suzuki, T. and Uemura, H. (1999). A human gene, hSGT1, can substitute for GCR2, which encodes a general regulatory factor of glycolytic gene expression in Saccharomyces cerevisiae. Mol. Gen. Genet. 260: 535-540. 9928932
Siviter, R. J., et al. (2002). Ance, a Drosophila angiotensin-converting enzyme homologue, is expressed in imaginal cells during metamorphosis and is regulated by the steroid, 20-hydroxyecdysone. Biochem, J. 367(Pt 1): 187-93. 12093364
Sliter, T. J. (1989). Imaginal disc-autonomous expression of a defect in sensory bristle patterning caused by the lethal(3)ecdysoneless1 (l(3)ecd1) mutation of Drosophila melanogaster. Development 106: 347-354. 2512110
Sorrentino, R. P., Carton, Y. and Govinda, S. (2002). Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev. Biol. 243: 65-80. 11846478
Thomas, S. R. and Lengyel, J. A. (1986). Ecdysteroid-regulated heat-shock gene expression during Drosophila melanogaster development. Dev. Biol. 115(2): 434-8. 3086161
Warren, J. T., Bachmann, J. S., Dai, J. D. and Gilbert, L. I. (1996). Differential incorporation of cholesterol and cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3)ecd1 mutation. Insect Biochem. Mol. Biol. 26: 931-943. 9014338
Warren, J. T., Wismar, J., Subrahmanyam, B. and Gilbert, L. I. (2001). Woc (without children) gene control of ecdysone biosynthesis in Drosophila melanogaster. Mol. Cell Endocrinol. 181: 1-14. 11476936
Warren, J. T., Petryk, A., Marques, G., Jarcho, M., Parvy, J. P., Dauphin-Villemant, C., O'Connor, M. B. and Gilbert, L. I. (2002). Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster. Proc. Natl. Acad. Sci. 99: 11043-11048. 12177427
Wismar, J., Habtemichael, N., Warren, J. T., Dai, J. D., Gilbert, L. I. and Gateff, E. (2000). The mutation without children (wocrgl) causes ecdysteroid deficiency in third-instar larvae of Drosophila melanogaster. Dev. Biol. 226: 1-17. 10993670
Zhang, Y., et al. (2006). The human orthologue of Drosophila Ecdysoneless protein interacts with p53 and regulates its function. Cancer Res. 66(14): 7167-75. 16849563
date revised: 20 December 2006
Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.