Ecdysone receptor

DEVELOPMENTAL BIOLOGY (part 2/2)

Larval and Pupal (part 2)

In larvae of the hawkmoth, Manduca sexta, accessory planta retractor (APR) motoneurons undergo a segment-specific pattern of programmed cell death at pupation. APR death is triggered hormonally by the prepupal peak of the ecdysteroid 20-hydroxyecdysone (20-HE). APRs were removed from the nervous system before the prepupal peak and placed in low density cell culture. Physiological levels of 20-HE trigger the same segment-specific pattern of APR death in vitro as seen in vivo. The presence or absence of contact with other cells does not influence the response of APRs to 20-HE. The death of APRs in culture is characterized by fragmentation or rounding up of the cll body and fragmentation of the neurites. These findings suggest that intrinsic segmental identity regulates whether these motoneurons live or die when exposed to a steroid hormone during development. Most Manduca motoneurons including the APRs express ecdysteroid receptors during the prepupal peak, consistent with the possibility of direct hormone action (Streichert, 1997).

One system that has proven amenable for the study of programmed cell death is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. These giant muscle cells are used during the eclosion (emergence) behavior of the adultmoth, and then die during the subsequent 30 h. For the ISMs, the trigger for PCD is a decline in the circulating titer of the insect molting hormone, 20-hydroxyecdysone (20-HE). During cell death there are rapid decreases in both the myofibrillar sensitivity to intracellular calcium and the resulting force of fiber contraction. The ability of the ISMs to undergo PCD requires the repression and activation of specific genes. Two of the repressed genes encode actin and myosin. One of the upregulated presumptive cell-death genes encodes polyubiquitin, which appears to play a critical role in the rapid proteolysis that accompanies ISM death (Schwartz, 1992).

The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event. The induction of polyubiquitin mRNA is accompanied by a proportional increase in total ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at eclosion, during which loss of muscle protein mass is maximum. A smaller but measurable increase in ubiquitin conjugates is observed earlier in pupal development coincident with a modestly enhanced degradation of myofibrillar proteins. Accumulation of ubiquitin conjugates is accompanied by induction in the pathway for polypeptide ligation, including the activating enzyme (E1), several carrier protein (E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both accumulation of ubiquitin polypeptide and the enzymes of the conjugation pathway are subject to regulation by declining titers of the insect molting hormone 20-hydroxyecdysone, which signals onset of programmed cell death in the intersegmental muscles. Thus, programmed cell death within the intersegmental muscles is accomplished in part by stimulation of the ubiquitin-mediated degradative pathway through a coordinated induction of ubiquitin and the enzymes responsible for its conjugation to yield proteolytic intermediates. This suggests enzymes required for ubiquitin conjugation may represent additional genes recruited for developmentally programmed death (Haas, 1995).

Ecdysteroids regulate the remodeling of the dorsal external oblique 1 (DEO1) muscle during metamorphosis in Manduca sexta. The temporal and spatial patterning of the A and B1 isoforms of the ecdysone receptor (EcR) within muscle DEO1 corresponds to the developmental fates of the fibers. Using antibodies directed to specific isoforms of EcR, it has been shown that the expression of various EcR isoforms in myonuclei differ among the five fibers of DEO1 and correspond to the developmental response of the muscle to the changing steroid titers and to the pattern of innervation. Muscle degeneration and apoptosis of myonuclei in all fibers are correlated with the expression of only EcR-A just before pupal ecdysis and then with the expression of low levels of both EcR-A and EcR-B1 shortly after pupation. Only the first fiber of muscle DEO1 participates in the regrowth of the adult muscle, and only this fiber shows an upregulation of EcR-B1 that is evident at 3 d after pupal ecdysis. Denervation of the muscle prevents both the upregulation of EcR-B1 and myoblast proliferation. It is concluded that the developmental fate of muscle DEO1 during metamorphosis is orchestrated by interactions between rising and falling ecdysteroid titers, the pattern of expression of EcR isoforms by the muscle, and interactions with other cells in the local environment (Hegstrom, 1998).

Proliferation zones in each optic lobe for EcR expression begin in the late second instar, expressing only the B1 isoform. Levels peak 24 hours prior to pupariation but by pupariation the optic lobe is devoid of EcR-B1. This expression may be related to the unique development of the optic lobe during larval life, correlated with development of the compound eye. During this period ingrowing retinal axons induce proliferation in the lamina. The gradient of axon ingrowth that ends about 10 hours after pupariation sets up in the lamina a corresponding gradient of differentiation that is evident through at least the next 30-40 hours (Truman, 1994).

Cell proliferation within the optic lobe anlagen is dependent on ecdysteroids during metamorphosis of the moth Manduca sexta. Cultured tissues were used to show that ecdysteroids must be maintained above a sharp threshold concentration to sustain proliferation. Proliferation can be turned on and off repeatedly simply by shifting the ecdysteroid concentration to levels above or below this threshold. In subthreshold hormone, cells arrest in the G2 phase of the cell cycle. Ecdysteroid control of proliferation is distinguished from differentiative and maturational responses to ecdysteroids by requiring tonic exposure to the hormone and lower levels of 20-hydroxyecdysone, and by being sensitive to either 20-hydroxyecdysone or its precursor, ecdysone. These characteristics allow optic lobe development to be divided into two ecdysteroid-dependent phases. Initially, moderate levels of ecdysteroid stimulate proliferation. Later, high levels of 20-hydroxyecdysone trigger a wave of apoptosis within the anlage that marks completion of its proliferative phase (Champlin, 1998a).

The optic lobe is composed of three ganglia, the lamina, medulla and lobula. The neurons of the optic ganglia are progeny of neuroblasts in the optic lobe anlage (OA). In Manduca sexta, the neuroblasts are arranged in a double-banded bracelet that comprises the inner and outer OA. During early larval stages, expansion of the neuroblast population occurs by symmetric cell divisions. In the final larval instar, the neuroblasts switch to the asymmetric divisions that lead to neuron production. The smaller daughter of each asymmetric division, the ganglion mother cell (GMC), divides to produce neurons. GMCs of the inner OA produce the neurons of the lobula. The medial margin of the outer OA is composed of the neuroblasts and GMCs that produce the neurons of the medulla (medulla precursor cells, MPC), while the lateral margin is composed of the neuroblasts and GMCs that produce the neurons of the lamina (lamina precursor cells, LPC) (Champlin, 1998a).

Proliferation of the LPC has been shown to be regulated by incoming retinal afferents. Transection of the optic nerve in Manduca also leads to disruption of LPC proliferation. Communication via retinal afferents provides a coordinating link between the developing retina and production of lamina neurons. Ecdysteroids, by contrast, control production of neurons throughout the optic lobe. The precursors for the medullar and lobular neurons both require ecdysteroids for proliferation. The optic nerve is severed in cultures making it difficult to determine if the same is true for the LPCs. Despite these conditions, arrested LPCs still enter mitosis in response to 20E. It appears that neural precursors throughout the optic lobe have a similar ecdysone-dependent G2 checkpoint. The failure of LPCs in cultured brains to incorporate BUdR suggests that 20E allows the cells to divide but they then arrest in G1 in the absence of innervation. This interpretation is consistent with data from Drosophila that LPCs arrest in G1 in mutants lacking proper retinal innervation. Thus, the cell cycle of LPCs in Manduca may have two developmentally regulated control points, stimulation by retinal afferents being required for progression through G1 and stimulation by ecdysteroids being required for progression through G2 (Champlin, 1998a).

MPCs are found to proliferate in an ecdysteroid-dependent manner, even in small pieces of the outer OA, clearly indicating that other long-range signals are not required for these cells. Cells throughout the OA, including the MPCs, express the ecdysteroid receptor; therefore, these cells could be responding directly to ecdysteroids. An important issue is whether the ecdysteroid-dependent proliferation of the MPC is a direct response to the steroid or due to short-range signals from neighboring cells. Evidence for communication between surrounding glia and neuroblasts is seen for the protein product of the anachronism locus, which is secreted by nearby glial cells and inhibits proliferation of optic lobe neuroblasts in Drosophila. The fact that the MPCs appear to respond to ecdysteroid as a unit suggests that there may be coordinating signals within the OA of Manduca (Champlin, 1998a).

Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. Genetic interaction and phenotypic analysis is presented showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain (Park, 2001).

Several genes have been identified that affect neuroblast proliferation, including anachronism (ana), terribly reduced optic lobes (trol) and eve. trol was originally identified in a genetic screen for abnormal larval brain morphology that was due to defective patterns of neuroblast proliferation in the larval brain. Mutations in trol cause a dramatic decrease in the reactivation of proliferation from mitotic quiescence. Recent studies suggest that trol may regulate this reactivation of neuroblast proliferation by stimulating the G1/S transition through upregulation of Cyclin E (CycE) expression. Several studies on trol and ana have led to the hypothesis that trol is required to overcome the repression of neuroblast cell division imposed by ana. eve was identified in a screen for enhancers of a hypomorphic allele trol. Mutations in eve enhanced both the trol proliferation phenotype and the associated lethality, indicating that eve may regulate transcription of cell cycle genes in the trol pathway (Park, 2001).

Analysis of explants has shown that ecdysone enables activation of neuroblast division and can substitute for larval extract. Furthermore, addition of ecdysone does not rescue the proliferation phenotype of cultured trol mutant brains, implying that ecdysone acts upstream of trol. Thus, ecdysone can overcome the lack of eve-induced activity in extracts of mutant flies. Interestingly, almost complete rescue is obtained when animals are fed ecdysone from 16-20 hours posthatching, indicating that the time between ecdysone action and S phase entry is at most four hours (Park, 2001).

The genetic interaction between eve and trol has all the characteristics expected for two components of a common pathway: (1) the eve;trol interaction is not allele specific and the known functional domains of Eve are implicated in the interaction; (2) the strength of the interaction mirrors the strength of the eve allele in segmentation; (3) eve mutants themselves have the predicted proliferation phenotype; and (4) neuroblasts arrested in trol;eve double heterozygotes can be rescued by expression of CycE, as can the neuroblasts arrested in a strong trol mutant. The latter is especially revealing, as induction of CycE expression in trol mutants results in the activation of cell division only in the number of neuroblasts appropriate to the developmental stage of the induction. That is, not all mitotically quiescent neuroblasts are arrested at the same cell cycle phase, and the extent to which CycE is a limiting factor is developmentally controlled. Therefore, as in embryonic segmentation and determination of neuronal identity, eve appears to function in a specific genetic pathway to affect the behavior of specific cells at specific times (Park, 2001).

However, Eve is not detectable in regulated neuroblasts at any time during first instar. Furthermore, eve function is not required within the larval CNS, but is required within the larval body from which extracts are prepared. Moreover, low levels (10%-20%) of extract made from eve plus animals will not support activation of neuroblast division while higher concentrations will. This concentration dependence indicates that eve does not inhibit production of a trans-acting proliferation repressor that is produced at higher levels in a eve mutant, since dilution of such a repressor would allow neuroblast division at lower rather than higher extract concentrations. These results strongly suggest that eve function is required for the production of a trans-acting factor that stimulates neuroblast division (Park, 2001).

Is ecdysone the trans-acting factor produced in response to eve? Ecdysone can rescue eve-dependent proliferation defects both in vivo and in vitro, but not the proliferation defect of trol mutants in vitro. This suggests that ecdysone acts upstream of trol, as would be expected if it is the eve-dependent trans-acting signal, and trol acts within the receiving cells. However, while the ecdysone receptor has been detected in a few neurosecretory cells of the first instar CNS, it has not been detected in neuroblasts. This may indicate that only a few high-affinity receptors are required to transduce the ecdysone signal, or that ecdysone acts indirectly through the products of the neurosecretory cells. However, as Eve is not detectable in the neurosecretory cells in wild-type brain lobes, it is unlikely that the added ecdysone rescues mutant animals by compensating for a loss of Eve activity in those cells. In each of these cases, eve could be acting through ecdysone production. Alternatively, ecdysone may act through a parallel pathway to that stimulated by an (unknown) eve-dependent signal. While the relationship between eve and ecdysone is not yet clear, it seems likely that eve is required for the production of an organismal-level trans-acting signal that is specifically required to stimulate larval neuroblast proliferation (Park, 2001).

The eye primordium of the moth, Manduca sexta, shows two different developmental responses to ecdysteroids depending on the concentration to which it is exposed. Tonic exposure to moderate levels of 20-hydroxyecdysone (20E) or its precursor, ecdysone, are required for progression of the morphogenetic furrow across the primordium. Proliferation, cell-type specification and organization of immature ommatidial clusters occur in conjunction with furrow progression. These events can be reversibly started or stopped in cultured primordia simply by adjusting the levels of ecdysteroid either above or below a critical threshold concentration. In contrast, high levels of 20E cause maturation of the photoreceptors and the support cells that comprise the ommatidia. Ommatidial maturation normally occurs after the furrow has crossed the primordium, but premature exposure to high levels of 20E at any time causes precocious maturation. In such cases, the furrow arrests irreversibly and cells behind the furrow produce a well-formed, but miniature, eye (Champlin, 1998b).

To sustain furrow progression in culture, the concentration of 20E had to be maintained between 1.2x10 -7 M and 2x10 -6 M. Outside of this range, little or no BUdR incorporation is found in cells flanking the furrow, even when the labeling period is extended through 24 hours. In contrast, when eye primordia were cultured with 20E concentrations within the 'proliferative' range, a high number of S phase and M phase cells, comparable to that observed in freshly dissected primordia are detected flanking the furrow. Their frequency appears to be fairly constant regardless of whether primordia are cultured at the high or low end of the proliferative range. To confirm that the furrow was indeed advancing in culture, primordia were exposed to a second pulse of BUdR 13 hours after the first. The distance between cells labeled by the two pulses of BUdR is similar regardless of whether primordia are cultured in 20E concentrations at the low or high end of the proliferative range, showing that the rate of furrow progression is independent of steroid concentration within this range. Both ends of the proliferative range show sharp thresholds beyond which the furrow is arrested. The type of arrest, though, is different for the lower and upper ends of the range. The concentration causing 50% arrest (ED50) for the lower threshold is about 1.2x10 -7 M concentration of 20E; this is similar for eye primordia isolated either from P+1 or P+4 day animals. Furrow progression stops in concentrations of 20E below this threshold, but resumes any time the 20E concentration is shifted up into the proliferative range. In contrast, the furrow arrest caused by concentrations of 20E above the proliferative range is not reversed when the concentration of 20E is shifted down into the proliferative range, even though the furrow has traversed only a portion of the eye primordium (Champlin, 1998b).

Ecdysone, the precursor for 20E, is widely regarded as an inactive prohormone. However, it was noted that the furrow continues advancing early in the pupal stage when ecdysone is essentially the only ecdysteroid detected in the blood. Ecdysone is also able to maintain furrow progression in culture at concentrations above a threshold of 6x10 -7 M, a concentration about fivefold higher than the threshold for 20E. However, in contrast with 20E, high concentrations of ecdysone do not cause furrow arrest even at the highest levels tested (Champlin, 1998).

Interruption of furrow progression during diapause (overwintering) Manduca larvae can be programmed by short-day length to enter a diapause state shortly after they pupate. Animals destined to diapause are indistinguishable from those programmed by long-day length for continuous development until P+2 days, and the morphogenetic furrow continues advancing through this time. Prothoracicotropic hormone, the neuropeptide that stimulates ecdysteroid synthesis, fails to be released in short-day animals on P+2 days: the ecdysteroid titer drops to low levels, and diapause ensues. By day P+3, furrow progression has ceased in short-day animals at a point about one-third of the way across the primordium. The morphogenetic furrow then remains arrested during the months that pupae are in diapause even though the animals may remain at the normal rearing temperature. Diapausing pupae can be induced to resume development at any time by injection of physiological doses of ecdysteroid. In such animals, incorporation of BUdR in cells flanking the furrow is detected within 12 hours of injection. The concentration of 20E needed to stimulate resumption of furrow progression in eye primordia isolated from pupae in diapause is identical to that needed to maintain furrow progression in primordia isolated from long-day animals (Champlin, 1998b).

Precocious and catastrophic metamorphosis occurs throughout animals treated with high levels of 20E, suggesting that ecdysteroids control development of other tissues in a manner similar to the eye. The threshold concentrations of 20E required for furrow progression versus ommatidial maturation differ by about 17-fold, with maturation requiring higher levels. This capacity to regulate distinct phases of development by different concentrations of a single hormone is probably achieved by differential sensitivity of target gene promoters to induction by the hormone-bound receptor(s). Expression of early genes was examined in eye primordia that were cultured with different levels of 20E in the presence of anisomycin, an inhibitor of protein synthesis. Different early genes are induced at concentrations that correspond to the thresholds for furrow progression and for ommatidial maturation, respectively. For example, MHR3 is the Manduca homolog of the Drosophila nuclear receptor DHR3, an early gene that plays an integral role in maturational responses to ecdysteroid. The induction of MHR3 mRNA requires high concentrations of 20E in the range needed to trigger maturation. In contrast, direct upregulation of the ecdysone receptor mRNA occurs at lower 20E concentrations, in the range that maintains furrow progression (Champlin, 1998b).

Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the Ecdysone receptor (EcR) and its partner Ultraspiracle (Usp). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of Usp and the EcR-A isoform in all but one of eight oocytic mesoderm-derived somatic cell types. The eight oocytic mesoderm-derived somatic cell types consist of apical cells, terminal filament cells, cap cells, the epithelial sheath, the inner germarial sheath cells, the follicle cells, the basal stalk and the oviduct. The first recognizable event of ovarian differentiation is the formation of the terminal filaments (TFs), a process of convergent extension that begins at around 12 hours after ecdysis to the third instar, and continues throughout the remainder of the final larval stage. At pupariation (the onset of the larval-pupal transition), all of the approximately 21 TF stacks have formed, and the location of these stacks prefigures the positions of the mature ovarioles (the functional units of the ovary). During TF differentiation, three additional cell types are present: germ cells (in the central region of the ovary); apical cells (anterior to the germ cells), and basal somatic cells (posterior to the germcells). At pupariation, a subset of the apical cells, the epithelial sheath cells, have begun to surround each terminal filament, and will ultimately separate the ovarioles. The epithelial sheath and the other apical cells are collectively referred to as anterior somatic cells. At pupariation, an additional somatic cell type is distinguishable: the cap cells occupy a position between the TF cells and the germ cells (Hodin, 1998).

By 12 hours after pupariation (12 h AP), the germ cells have begun to form cysts and the epithelial sheaths are continuing to advance in an apical to basal progression. Adjacent to the germ cells reside inner germarial sheath cells, which line the interior lateral edges of the germarium (the birth place of the egg chambers). At 12 h AP, the basal somatic cells remain in a predifferentiative state. By 18 h AP, follicle cells have begun to surround the first egg chambers. At 24 h AP, the epithelial sheaths have separated the ovaries into ovarioles and the basal somatic cells begin to differentiate into the basal stalks and the anterior oviduct (cells at the extreme posterior of the ovary that will eventually fuse with the genital disc-derived oviduct). By 42 h AP, the ovaries and oviducts have fused, and the basal stalks are present (Hodin, 1998).

The exceptional cell type, that is, the one in which EcR cannot be detected, is the larval TF cell, in which only Usp is detectable during cell differentiation. The onset of TF formation is accompanied by the onset of expression in the presumptive TF cells of the Bric-a-brac protein, which is required for proper TF formation. In cells destined to form the basal stalks and anterior oviduct, Usp colocalizes with what appears to be the EcR-B2 isoform. BrdU incorporation in pupal ovaries correlates with ecdysteroid levels, suggesting that ecysteroids regulate proliferation in the ovary, presumably via the EcR-Usp heterodimeric receptor. EcR-A is first detected at approximately 12 h after ecdysis into the third instar, in the somatic cells of the ovary but not in the forming TF cells. The EcR-A isoform is abundant only in the anterior cells (Hodin, 1998).

Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. In such mutants there is a significant increase in the number of TF cells formed (identified by Bric-a-brac expression), but there are fewer TF stacks. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Thus usp3 heterozygotes exhibit accelerated TF formation; the Df EcR heterozygotes show delayed TF formation, and the EcR-usp double heterozygotes are delayed both in the onset and in the completion of TF formation. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. These results demonstrate that proper expression of the Ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the Ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects. It is concluded that heterochronic shifts in ovarian differentiation have apparently been accomplished by uncoupling the process of ovarian differentiation from tissue differentiation in the rest of the animals and that alterations in timing of expression of the EcR complex may reside in a more general mechanism by which heterochronic changes in the differentiation of individual tissues have been accomplished in insect evolution (Hodin, 1998).

Adults

In many sexually mature insects, egg production and oviposition are regulated as consequence of copulation. . Sex-Peptide (SP) is a 36-amino-acid peptide synthesized in the accessory glands of Drosophila melanogaster males and is transferred to the female during copulation. Sex-Peptide stimulates vitellogenic oocyte progression through a putative control point at about stage 9 of oogenesis. Application of the juvenile hormone (JH) analog methoprene mimics the Sex-Peptide-mediated stimulation of vitellogenic oocyte progression in sexually mature virgin females. Apoptosis is induced by 20-hydroxyecdysone in nurse cells of stage 9 egg chambers at physiological concentrations [10(-7) M]. 20-Hydroxyecdysone thus acts as an antagonist of early vitellogenic oocyte development. However, simultaneous application of JH analog protects early vitellogenic oocytes from 20-hydroxyecdysone-induced resorption. These results suggest that the balance of these hormones in the hemolymph regulates whether oocytes will progress through the control point at stage 9 or undergo apoptosis. These data are further supported by a molecular analysis of the regulation of yolk protein synthesis and uptake into the ovary by the two hormones. It is concluded that JH is a downstream component in the Sex-Peptide response cascade and acts by stimulating vitellogenic oocyte progression and inhibiting apoptosis. Since juvenile hormone analogue does not elicit increased oviposition and reduced receptivity, Sex-Peptide must have an additional, separate effect on these two postmating responses (Soller, 1999).

SP stimulates JH biosynthesis in corpus allatum complexes isolated from sexually mature virgin females in vitro. Consistent with this finding, JH application stimulates progression of oocytes through the control point at stage 9, involving an increased uptake of from the hemolymph and an increased synthesis of yolk proteins in the ovary. JH also protects early vitellogenic oocytes from ecdysone-mediated resorption. Thus, after mating, ecdysone-mediated oocyte resorption in virgins is relieved due to the increase of JH levels. The corpus allatum is likely to be a target organ for SP action. Since application of JH neither induces a reduction in receptivity nor elicits complex behavioral change, neuronal tissues have to be considered further targets of SP (Soller, 1999 and references).

The similarity of Taiman to steroid hormone receptor coactivators suggests that Tai might interact with one or more steroid hormone receptors. The only known steroid hormone in Drosophila is ecdysone, and the ovary is a major site of ecdysone synthesis, which peaks at stage 9. The functional ecdysone receptor is a heterodimer composed of Ultraspiracle (Usp), which is the fly retinoid X receptor (RXR) homolog, and the Ecdysone receptor. To determine whether the ecdysone receptor complex would be a good candidate for interaction with Tai, expression of ecdysone receptor subunits in egg chambers was examined using antibodies against Usp, EcR-A, and EcR-B. EcR-A and EcR-B are distinct isoforms of the EcR subunit, which are generated by alternative splicing. Usp, EcR-A, and EcR-B colocalize with Tai protein in migrating border cells; Usp and EcR-A are expressed generally, in both follicle cells and nurse cells (Bai, 2000).

These observations raise the possibility that the timing of border cell migration might be controlled by ecdysone. To test whether border cell migration is responsive to hormone, the effects of injecting hormone into female flies were examined. It was not expected that increasing the hormone concentration alone would be sufficient to cause precocious border cell migration because expression of the slbo gene and its targets are independently required for migration. Therefore, slbo was precociously expressed using transgenic flies carrying a heat-inducible slbo transgene, followed by injection of hormone. Border cell migration was assayed in stage 8 egg chambers dissected from flies treated with heat shock and hormone, and compared to control flies treated with heat shock and ethanol, or with hormone in the absence of heat shock. Precocious border cell migration was observed in 20% of egg chambers that were treated with both heat shock and hormone but not in controls. The observed effects are consistent with a role for ecdysone in regulating the timing of border cell migration (Bai, 2000).

If the rising ecdysone level at stage 9 is required to stimulate border cell migration, then reducing the ecdysone level should cause a delay in border cell migration. The ecdysoneless mutant ecd¹ is temperature sensitive for production of ecdysone. Females homozygous for ecd¹ are sterile when held at the nonpermissive temperature for 5 days, and egg chambers in these flies arrest development at stage 8 and subsequently degenerate. Border cells fail to develop in these arrested egg chambers. However, when ecd¹ mutants are held at the nonpermissive temperature for 2 days, some stage 10 egg chambers develop, in which border cells differentiate and express Slbo protein. Greater than 50% of these egg chambers exhibit delayed border cell migration (Bai, 2000).

Since the effects on border cell migration of increasing or decreasing ecdysone levels could have been indirect, whether there is a cell autonomous requirement for the ecdysone receptor in border cells was tested. The EcR locus is proximal to available FRT insertion sites, preventing mosaic analysis. Therefore, the analysis was carried out using mutations in usp. Border cells that were homozygous mutant for a null allele of usp exhibit inhibition of border cell migration, but no obvious defects in other follicle cells (Bai, 2000).

To assess whether Tai and the ecdysone receptor are likely to associate in a complex in vivo, Tai expression was examined in third instar larvae. Antibodies against Tai react specifically with the salivary gland nuclei, as well as other larval tissues. Polytene chromosome spreads were stained with antibodies against Tai and Usp proteins in a double labeling experiment. Anti-Tai antibody labels specific loci on the polytene chromosomes. Moreover, Usp and Tai proteins colocalize precisely. Since previous experiments have shown that Usp and EcR colocalize as a complex on polytene chromosomes, these results indicated that Tai colocalizes with the functional Ecdysone receptor complex at specific target sites (Bai, 2000).

Whether expression of Tai can enhance ecdysone receptor-dependent transcriptional activation in EcR-293 mammalian cells was tested. These cells respond to hormone, either ecdysone or an analog known as ponasterone, with a substantial increase in transcriptional activation of genes placed under the control of a cis-acting sequence known as an E/GRE. Transcriptional activation was tested in cells expressing varying amounts of Tai in transient transfection assays. Tai expression increases transcriptional activation up to 5-fold, in a dose-dependent manner, specifically in the presence of hormone (Bai, 2000).

Furthermore, a GST-fusion protein containing the region of Tai protein containing the LXXLL motifs predicted to interact with EcR (residues 1028 to 1235 of Tai) associates with in vitro translated EcR in a ligand-dependent manner. The same fusion protein does not associate detectably with Usp alone. However, in the presence of EcR and ligand, the Tai-GST fusion protein is able to coprecipitate Usp. Taken together, these results suggest that Tai is a bona fide ecdysone receptor coactivator (Bai, 2000).

Thus, Tai appears to be a coactivator of the p160 class based not only on amino acid sequence similarity and overall domain structure, but based also on its in vivo colocalization with EcR, its direct, ligand-dependent binding to EcR, and its ability to potentiate hormone-dependent transcription in cultured cells. The homology of Tai to SRC proteins suggests that Tai might interact with a steroid hormone receptor. Although there are more than 20 genes in Drosophila that code for proteins related to nuclear hormone receptors, ecdysone is the only known steroid hormone. Since SRC proteins require the presence of a ligand in order to interact with receptors, the ecdysone receptor seems like the best candidate partner for Tai. The colocalization of Tai protein with the ecdysone receptor complex at specific chromosomal loci in third instar larva, the direct and ligand-dependent binding of Tai to EcR in vitro, and the ability of Tai to potentiate the ecdysone response in cell culture lend substantial support to this proposal (Bai, 2000).

The ligand-dependent interaction of Tai with the ecdysone receptor suggests that ecdysone regulates border cell migration. The strongest evidence in support of this is that border cells lacking Usp are unable to migrate. Consistent with this observation, numerous unfertilized eggs were produced from females lacking usp function. Moreover usp is required specifically in somatic cells for production of a fertilizable egg. Defects in border cell migration are known to lead to the production of unfertilized eggs. Whether EcR loss of function mutations affect border cell migration could not be examined. This is because the EcR locus, at 42A, is proximal to available FRT insertions, making it impossible to make FLP-mediated mosaic clones. The frequency of X-ray induced mitotic clones is too low to be useful, and marking such clones is problematic. A temperature-sensitive allele of EcR exists and flies at the nonpermissive temperature exhibit a variety of defects in oogenesis, including arrest prior to border cell migration. Even though it was not possible to assess the effect of EcR mutations specifically in the border cells, the observations that hormone injections can lead to precocious border cell migration and that reduced ecdysone levels can lead to delayed migration provide additional support for the hormonal control of migration (Bai, 2000).

The rise in ecdysone after eclosion, specifically in females, occurs in response to adequate nutrition. In the absence of a rich diet, yolk protein synthesis is inhibited and oogenesis does not progress. Yolk protein synthesis can be restored in the absence of a rich diet by applying ecdysone or juvenile hormone (JH) to cultured ovaries. Recent studies indicate that functional ecdysone receptors are required in the germline for progression of oogenesis through vitellogenesis, the stages during which yolk is taken up by the oocyte. In summary, then, adequate nutrition appears to lead to elevated hormone levels, which in turn stimulate yolk protein synthesis and uptake, and progression of oogenesis beyond stage 8. Together with the results reported here, these findings suggest that a rising ecdysone titer coordinates a variety of events that occur in early vitellogenic egg chambers, including border cell migration (Bai, 2000).

Nutritional status affects 20-hydroxyecdysone concentration and progression of oogenesis in Drosophila

Drosophila egg production depends upon the nutrition available to females. When food is in short supply, oogenesis is arrested and apoptosis of the nurse cells is induced at mid-oogenesis via a mechanism that is probably controlled by ecdysteroid hormone. Expression of some ecdysone-response genes is correlated with apoptosis of egg chambers. Moreover, ecdysteroid injection and application of juvenile hormone respectively induces and suppresses the apoptosis. In this study, an investigation was carried out to see which tissues show increases in the concentration of ecdysteroids under nutritional shortage to begin to link together nutrient intake, hormone regulation and the choice between egg development or apoptosis made within egg chambers. Ecdysteroid levels in the whole body, ovaries and haemolymph samples were measured by RIA, and it was found that the concentration of ecdysteroid increased in all samples. This contributes to the idea that nutritional shortage leads to a rapid high ecdysteroid concentration within the fly and that the high concentration induces apoptosis. Low concentrations of ecdysteroid are essential for normal oogenesis. It is suggested there is threshold concentration in the egg chambers and that apoptosis at mid-oogenesis is induced when the ecdysteroid levels exceed the threshold. Starvation causes the ovary to retain the ecdysteroid it produces, thus enabling individual egg chambers to undergo apoptosis and thus control the number of eggs produced in relation to food intake (Terashima, 2005).

The prothoracic glands, which are the principal source of ecdysone in the immature stages, are no longer present in adults. The egg chambers produce ecdysone, which, at least in some insects, accumulates in the oocyte. In the fat body, ecdysone is converted to 20E, the active hormone, and shade, which encodes 20-hydroxylase for converting ecdysone to 20E, is expressed in nurse cells and follicle cells in the ovary and fat body (Terashima, 2005).

Ecdysteroid synthesis is affected by the nutritional status of the female, and ecdysteroids affect oogenesis in many insects. Egg production in mosquitoes is triggered by a blood meal. The digested products of the blood meal stimulate the brain to secrete egg development neurosecretory hormone (EDNH), which is also known as ovarian ecdysteroidogenic hormone (OEH). EDNH stimulates the ovary to synthesize ecdysteroids, which instruct the fat body cells to make vitellogenin for the oocytes. Vitellogenin is critical for egg production, thus without the blood meal there is no vitellogenin and no eggs, so to produce mature eggs ecdysteroids are essential. In contrast, nutritional shortage induces an increase in ecdysteroid concentration in Drosophila females, ecdysteroid concentration increases in Drosophila whole body, haemolymph and ovaries during starvation. Feeding suppresses the high ecdysteroid concentration that is induced by nutritional shortage (Terashima, 2005).

Under starvation, apoptosis of nurse cells in stage-8 and -9 egg chambers is induced; 20E injection into the females under adequate nutrition also induces the apoptosis and JHA treatment of females under nutritional shortage suppresses this apoptosis. Presumably high ecdysteroid concentrations in the haemolymph and/or the ovary, which are induced by starvation, may induce the apoptosis of nurse cells in stage-8 and -9 egg chambers. However, ecdysteroid is indispensable to produce mature eggs in Drosophila. Oogenesis in ecd-1 mutants is arrested at mid-oogenesis, and germline clones of EcR mutations led to developmental arrest; egg chambers degenerated during mid-oogenesis in Drosophila. Presumably, there is an ecdysteroid threshold for inducing apoptosis of nurse cells at stages 8 and 9 and ecdysteroids induce normal development when below the threshold concentration and induce apoptosis of nurse cells at stages 8 and 9 when over the threshold. Starvation induces an increase in ecdysteroid concentration to above the threshold level in the haemolymph and the ovary through activation of the ecdysone synthesis pathway in the egg chamber. Ecdysteroid secretion from the ovary decreased following nutritional shortage. Thus, ecdysteroid secretion from the fat body or other ecdysteroid-synthesizing tissues must be stimulated to induce the high ecdysteroid concentration observed in haemolymph (Terashima, 2005).

JHA suppresses the high ecdysteroid concentration that is induced by starvation. JH and JHA suppress ecdysone synthesis/secretion from the prothoracic glands in larvae of Maduca sexta. It is likely that JHA suppression decreases the high ecdysteroid concentration in the ovary that induces apoptosis of nurse cells in stage-8 and -9 egg chambers under starvation, and therefore JHA treatment retains minimal ecdysteroid levels needed for inducing normal oogenesis (Terashima, 2005).

There is a developmental checkpoint at stage 8 of oogenesis. YP synthesis commences at stage 8 and YP is accumulated during development into mature eggs. Drosophila egg chambers normally transit through stages 8 and 9 during a 6-h period, but starvation induced an accumulation of stage-8 and -9 egg chambers in Drosophila oogenesis. The number of stage-8 egg chambers is increased during a 5-12-h period after starvation starts, but the number of stage-9 egg chambers does not increase for 0-12 h after starvation started. This means that oogenesis progresses from stage 7 to 8, but does not progress from stage 8 to 9 and then to 10 under nutritional shortage. When 20E is injected into the fed flies, the accumulation of stage-8 chambers is not seen; therefore this arrest of oogenesis at stage 8 was not caused by the increasing 20E concentration in haemolymph and ovary. Perhaps starvation signals induce the arrest of oogenesis at stages 8 and 9 directly, or they could inhibit YP uptake. Some nutrient- and stress-response genes exhibit different expression patterns in the ovaries of females under adequate nutrition and starvation. It is suggested that the genes which respond directly to stress and nutrients interact with the ecdysone-synthesis pathway, resulting in the induction of apoptosis of nurse cells in stage-8 and -9 egg chambers through activation of BR-C Z2, Z3 and E75A expression in the follicle cells. Other genes could have altered their expression levels, so as to arrest oogenesis at stages 8 and 9 and to check the developmental status of the egg chamber. As a result, the decision is made to develop into a mature egg or undergo apoptosis at stages 8 and 9. The arrest in the progression of oogenesis at stages 8 and 9 is independent of increasing ecdysteroid levels (Terashima, 2005).

Starvation signals are needed to activate a number of pathways to adjust the rate of egg production in Drosophila. These pathways could be classified into two groups: one to stimulate ecdysone synthesis in the follicle cells and/or nurse cells to activate the apoptosis pathway, including BR-C Z2, Z3 and E75A expression in the follicle cells, and another one to interact with and participate in the developmental checkpoint, giving rise to an arrest in oogenesis at stage 8 under nutritional shortage. A possible scheme is presented for the regulation of oogenesis related to nutrition in Drosophila. It is likely that starvation signals from the gut activate ecdysteroid synthesis in the ovary in Drosophila under starvation. Ecdysteroid is then accumulated in the egg chamber by decreasing 20E secretion from the ovary, and the fat body secretes 20E to haemolymph. It is suggested that there are two thresholds of 20E concentration in Drosophila ovary -- one is the concentration for normal oogenesis and the other is the concentration for inducing apoptosis -- and that starvation elevates the ecdysone levels in some egg chambers over the threshold that leads to apoptosis (Terashima, 2005).

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