Ecdysone receptor


Effects of Mutation or Deletion

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three Ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. Two classes of EcR mutations have been identified and molecularly mapped: those specific to EcR-B1, which uncouple metamorphosis, and embryonic-lethal mutations, which map to common sequences encoding the DNA- and ligand-binding domains. The two mutations that are specific to the EcR-B1 isoform both generate stop codons that lie within the EcR-B1-specific exon 2 and are predicted to produce EcR-B1 proteins truncated after only 49 or 52 amino acids. Because neither mutation interferes with the synthesis or structure of EcR-A or EcR-B2, they provide a means for determining EcR-B1-specific functions. The EcR early mutants form a largely normal cuticle, which bears the proper number of ventral denticle belts. However, a number of more subtle defects are characteristic of these mutants, including reductions in denticle size and number, and occasional mouthpart aberrations. It should be noted that a maternal contribution of EcR-A mRNA and protein has been observed; hence, the phenotype of a full null EcR is apt to be more severe (Bender, 1997).

The EcR nonpupariating and prepupal mutant classes affect the early stages of metamorphosis, which commences with pupariation and the formation of the pupal case. Pupariation is preceded by a cessation of wandering and includes shortening of the larva, eversion of anterior spiracles, attachment to a solid surface, and hardening of the cuticle. The two EcR-B1 nonpupariating class mutants initiate wandering but fail to evert their spiracles, or to shorten and attach themselves to a solid surface, or to harden their cuticles. Despite failure of these early events, these mutants easily slip free of the larval cuticle when dissected, indicating that apolysis of the larval cuticle has been completed, and hence, that coordination of the early events of metamorphosis is disrupted. EcR-B1 is the predominant isoform in both imaginal and larval cells of the larval midgut; indeed, the imaginal cells do not express detectable EcR-A. Large polyploid cells of the larval epithelial layer line the lumen of the midgut. Interspersed with these larval cells are the midgut imaginal islands, small groups of diploid cells that will give rise to the adult gut. Within 2 hours after puparium formation, the cells of the midgut imaginal islands begin to proliferate. Ultimately the islands join to form a complete larval tube.

Neither cell type follows its normal developmental pathway at metamorphosis in EcR-B1 mutants. The imaginal cells of the midgut islands begin to proliferate but fail to form a tube surrounding the larval epithelial cells, and the larval epithelial cells fail to become condensed into a compact mass. The abdominal histoblasts, which give rise to the adult abdomen, express EcR-B1 but no detectable EcR-A. Counts of histoblast numbers in EcR-B1 mutants reveal that histoblast proliferation is initiated in these animals, but no replacement of larval cells is observed. Histoblast proliferation is limited to one or two cell doublings in these mutants. The BR-C, E74 and E75 early genes are submaximally or negligibly induced in the EcR-B1 mutant, relative to wild type. Transgenic expression of EcR-B1 in EcR-B1 mutant glands results in full repair of that loss. Expression of EcR-B2 isoform only partially restores the loss of EcR-B1, while expression of EcR-A isoform fails to restore the loss of EcR-B1 (Bender, 1997).

The expression of two Ecdysone receptor isoforms early in metamorphosis correlates with the ongoing developmental responses, and not with the developmental origin of the neuron cells. Thus, EcR-B1 is expressed predominantly in neurons undergoing regressive responses whereas EcR-A is predominant in neurons showing maturational responses. Although correlative in nature, these observations are consistent with the hypothesis that EcR-A and EcR-B1 trigger specific cellular responses during CNS metamorphosis. The recent isolation of EcR-B1 specific mutations has allowed tests of EcR-B1 functions in directing specific responses to ecdysteroids (Schubiger, 1998).

In contrast to mutations in EcR common exons, which inactivate all EcR isoforms and lead to embryonic lethality, mutations in an EcR-B1 specific exon result in a delayed death, which allows for larval viability, but lethality at the onset of metamorphosis. Loss of both EcR-B isoforms leads to an earlier death, predominantly at the first and second larval molt. The predominant time of death of EcR-B mutants is during the 1st and 2nd larval stages. The EcR-B lethal period can be contrasted with the strong embryonic lethality of EcR common exon mutations that inactivate all three EcR isoforms. The EcR-B lethal period also differs from that seen for an EcR-B1 stop codon mutant, since the EcR-B1 mutant shows little reduction in 2nd larval stage viability and has substantially greater numbers of surviving 3rd instar larvae than any of the EcR-B mutants. Thus the EcR-B lethal phase differs from that of both EcR null and EcR-B1 specific mutants. The EcR-B animals that arrest during larval development show defects in the process of larval molting. Animals arrested prior to the 2nd larval stage carry both 1st and 2nd larval mouth hooks, while those arrested prior to the 3rd larval stage carry both 2nd and 3rd larval mouth hooks (Schubiger, 1998).

A small fraction of the EcR-B mutants survive to the third larval stage. These animals feed and later come to the surface of the food, but do not wander up the sides of the vial, indicating that normal wandering behavior is affected. They then become sluggish and eventually immobile. Wild-type larvae empty their gut at the end of the wandering period, shortly before the animals contract and form the barrel-shaped puparium. The mutant animals empty their gut but do not contract or form a tanned puparium. In addition, the gas bubble that normally forms at about 3 hours after pupariation is not observed in EcR-B mutants. Nevertheless, some tissues in these arrested larvae continue to show developmental changes: the larval cuticle apolyzes and a large space between the cuticle and the epidermis appears at the anterior and posterior ends, although the tracheal trunks remain attached to the larval cuticle. Later, the mouth hooks are expelled, a process that normally occurs at 12 hours after pupariation, at the time of head eversion. It is evident that a new cuticle, presumably the pupal cuticle, has been secreted. Elongation of the imaginal discs in mutant animals begins normally up to a stage comparable to about 4 hours after pupariation. At this time the peripodial epithelium contracts in wild-type animals to allow the discs to evert to the exterior. In the mutants this contraction apparently does not occur and the uneverted discs continue to elongate, but within vesicles inside of the body. The appendage can then break through the peripodial membrane and elongate inside the body. In normal development the beginning of muscle histolysis is recognizable at 3 hours after pupariation by a loss of birefringence in the set of larval abdominal muscles that are histolyzed during the prepupal stage. In the mutants even at the time of mouth hook detachment the larval muscles are present and have retained their birefringence. In the gut, the larval gastric caecae (which normally histolyze during the prepupal stage) have shortened but are still present at the time of the developmental arrest (Schubiger, 1998).

At the onset of metamorphosis larval neurons lose their synaptic targets and begin to prune back their larval dendritic and axonal arbors. This first step in the remodeling process is preceded by high levels of EcR-B1 expression. Later during pupal development EcR-A is the predominant isoform expressed in these neurons; it is during this stage that adult-specific processes and synapses are made. From the correlation of the EcR expression patterns with the ongoing developmental changes, a functional role is thought to exist for the two EcR isoforms. For example, process regression is thought to be under the control of EcR-B1, whereas maturation is thought to be induced by EcR-A (Schubiger, 1998).

The null mutations for the EcR-B isoforms allow a test for a functional role of EcR isoform types. The investigation focussed on a small set of larval neurons that contain a peptide that cross-reacts with an antibody against the molluscan small cardio-active peptide B (SCP). These neurons are a subset of the FMRFamide expressing neurons. The SCP antibody strongly labels the neurons and reveals a characteristic projection pattern that can easily be followed as these larval neurons initiate remodeling. EcR-B1 is expressed by SCP labeled neurons in control white puparia, and these neurons, as well as many surrounding larval neurons, express EcR-B1 at the time of pupariation. The EcR-B mutants show no detectable EcR-B1 immunoreactivity, whereas the neurons of controls and mutants at the same stage both express EcR-A. The SCP labeling pattern in the ventral nerve cord of control 0 hour prepupa is distinguished by a prominent pair of ventral neurons in each thoracic neuromere (the Tv neurons) and several other neurons in the subesophageal ganglion. The neurites from these cells form a characteristic projection pattern that is particularly dense in the area of the subesophageal ganglion, forming a posteriorly directed branch. Shortly after pupariation the dendritic arbors begin to regress. By 5-6 hours after pupariation the SCP label is clearly reduced, and at the time of head eversion few dendrites remain (Schubiger, 1998).

The loss of labeled dendrites is thought to be caused by the regression of the dendritic arbors, rather than the loss of the neuropeptide from the finer processes. To test this interpretation the bovine microtubule-associated protein tau was expressed in a subset of the SCP-IR neurons. Using the GAL4-UAS system, GAL4 constructs were made with upstream sequences of the FMRFamide gene to drive UAS-tau in the Tv cells. Examination of the Tau pattern at pupariation and late prepupal stages reveals a clear reduction of processes in the older animals. This indicates that the reduced SCP labeling pattern seen as animals metamorphose reflects process regression and not loss of neuropeptide from neuronal processes. In the EcR-B mutants the SCP pattern, at a stage closely corresponding to pupariation, is indistinguishable from the control, with a dense mesh of dendrites in the anterior region of the ventral CNS. In the mutants that develop to the stage of mouth hook detachment, a stage comparable to head eversion, the SCP pattern remains larval, despite the clear advancement of CNS development. Process regression does not occur and the dense dendritic arbors typical of younger stages are still present. The lack of pruning in older stage animals is consistent, and typical for all alleles tested, either in combination with an EcR null mutation or as homozygotes. These results indicate that the EcR-B isoforms have an essential role in the pruning back process during neuronal remodeling. Since both EcR-B isoforms are eliminated in these mutants the pruning back cannot be ascribed to one or the other EcR-B isoform. Consequently, the SCP pattern was examined in a EcR-B1 specific mutant. Most of these mutant larvae reach the third instar and have a phenotype similar to the late stage arrested animals of other EcR-B mutants. At the time of mouth hook detachment clear pruning back has taken place. In all CNS preparations extensive process regression is seen, though the degree is variable, with patterns similar to controls between 6 hours after pupariation and head eversion. It is thought that blockage of neuronal process regression in EcR-B but not EcR-B1 mutants indicates functional redundancy of the EcR-B1 and EcR-B2 isoforms during dendritic pruning of SCP-IR neurons. Such apparent functional redundancy has been seen frequently in RAR knockout experiments in the mouse, with milder defects than expected if generated by loss of a single isoform (Schubiger, 1998).

Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, a genetic mosaic screen was conducted and one ultraspiracle (usp) allele defective in larval process pruning was discovered. Consistent with the notion that Usp forms a heterodimer with the Ecdysone receptor (EcR), it was found that the EcR-B1 isoform is specifically expressed in the MBgamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/Usp targets are dispensable for MB neuronal remodeling. This study demonstrates cell-autonomous roles for EcR/Usp in controlling neuronal remodeling, potentially through novel downstream targets (Lee, 2000).

The recently established MARCM (for mosaic analysis with a repressible cell marker) genetic mosaic system has allowed the study of functions of genes in various neural developmental processes in the Drosophila brain. The MARCM system allows unique labeling of homozygous mutant cells in a mosaic tissue, which is important for phenotypic analysis of individual mutant neurons in the complex brain. Because typical neurogenesis involves the generation of ganglion mother cells (GMCs) from neuroblasts followed by the formation of two postmitotic neurons from each GMC, the MARCM system can be used to mark the entire axonal and dendritic projections of single neurons if mitotic recombination occurs during GMC division (Lee, 2000 and references therein).

MARCM-based analysis has elucidated the cellular basis for the development of the mushroom bodies (MBs), including neuronal reorganization of the MB during metamorphosis. The MBs are prominent neuropils of the central brain that are essential for several forms of learning and memory. Each MB is composed of approximately 2,500 neurons, which are derived from four neuroblasts that undergo hundreds of asymmetric divisions through embryonic, larval, and pupal stages. Unlike most larval-born neurons, which are arrested as immature neurons until the pupal stage, MB neurons elaborate axonal and dendritic projections shortly after mitosis. In the larval brain, every MB neuron extends a single process from which dendrites branch out into the calyx. The axon extends further and then bifurcates into two major branches, one projecting medially and the other projecting dorsally. Interestingly, MB neurons generated prior to the mid-third instar stage, named gamma neurons, prune the medial and dorsal branches during early metamorphosis and subsequently project axons only into the medial gamma lobe of the adult MB. In contrast, the alpha'/beta' MB neurons that are born after the mid-third instar stage retain their larval projections during metamorphosis (Lee, 2000 and references therein).

Because the MARCM system further allows one to generate clones homozygous for any mutation of interest only in the uniquely labeled gamma neurons, the MB gamma neuron was used as a genetic model system to investigate the molecular mechanisms of neuronal remodeling. Both a forward genetic screen and a candidate gene approach have indicated that Usp, is essential for MB gamma neuron remodeling. The EcR-B1 isoform is specifically expressed in the MB neurons destined for remodeling, and it mediates the axonal pruning of MB gamma neurons independent of the surrounding cells. The individual functions of several ecdysone primary response genes, including Broad-Complex (BR-C), E74, and E75 were examined, and none of them were found to be essential for the EcR/USP-mediated MB remodeling. This study demonstrates cell-autonomous roles for EcR/USP in controlling MB neuronal remodeling, potentially through novel downstream targets (Lee, 2000).

Drosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis. Mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion: this indicates a role for ecdysone in these morphogenetic responses. In some homo- and hetero-allelic crol combinations a few adult escapers can be recovered. All of these escapers also display wing defects. Fifty-seven percent of the adult escapers have held out wings with a partial (blister) or complete (balloon) separation of the dorsal and ventral wing surfaces, while the remaining 43% have either malformed or completely unfolded wings. The blisters in crol mutant wings are generally large and do not appear to have sharp boundaries. These phenotypes indicate a role for crol in cell adhesion and wing morphogenesis, raising the possibility that ecdysone signaling might play a role in regulating these processes. To test this hypothesis, the role of the ecdysone receptor in wing development was analyzed using the hypomorphic EcRk06210 allele. The wings of EcRk06210 homozygous mutants display cell adhesion and morphogenetic defects similar to those seen in crol mutants. At 18°C, 24% of the eclosed adults display venation defects, 32% have malformed wings, and 13% display wing blisters. The blisters are usually small and centrally located, although blistering of the entire wing (a balloon wing) can occasionally be seen. The most frequent venation defects include a small, extra vein that originates from the third longitudinal vein; an additional anterior crossvein, and a 'delta' thickening at the intersection between the posterior crossvein and the fourth longitudinal vein. crol and EcR mutations are shown to interact with mutations in genes encoding the integrin subunits. The frequency of blisters in if mutants is enhanced three- to four-fold by crol1 and crol2 and approximately sevenfold by crol3. In contrast, no interaction was observed between mysnj42 and crol1 or crol2, whereas the frequency of blisters and balloons in mysnj42 mutants is increased three- to four-fold by crol3, and the frequency of balloon wings increased approximately sevenfold (D'Avino, 2000).

alpha-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of alpha- and beta-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. The expression patterns of alphaPS1 and alphaPS2 in culture appear almost identical. After ~1 h of culture in the presence of ecdysone, alphaPS1 and alphaPS2 transcript levels decrease rapidly, becoming very low by 8 h after hormone addition. The alphaPS3 (scab) gene consists of two transcription units [Long-alphaPS3 (L-alphaPS3) and Short-alphaPS3 (S-alphaPS3)] that initiate from different start sites. Interestingly, L-alphaPS3 mRNA accumulates rapidly in response to ecdysone, peaking by 6-8 h after hormone addition, while S-alphaPS3 transcription appears unaffected by the hormone. Similar to S-alphaPS3, betaPS mRNA levels remain uniform throughout the time course. Thus, only alpha-integrin subunits are regulated by ecdysone in cultured larval organs, and they are either induced or repressed in response to the hormone (D'Avino, 2000).

These findings suggest that altered integrin gene expression in crol and EcR mutants lead to the defects observed in wing morphogenesis and cell adhesion. However, integrins also function in a wide range of other biological pathways during development, including tissue morphogenesis, cytoskeletal reorganization, memory, and gene expression. These widespread functions raise the possibility that ecdysone-regulated integrin expression may control multiple events during metamorphosis. For example, the if V2 semilethal allele displays a misshapen leg phenotype that resembles the defective legs seen in crol mutants, indicating that alphaPS2 functions may be recruited by the ecdysone pathway to regulate leg morphogenesis. Furthermore, since alphaPS3 has been proposed to mediate synaptic rearrangements, its ecdysone-induced expression in late third instar larvae may contribute to the extensive neuronal remodeling that occurs in the central nervous system during metamorphosis. Further studies of the tissue-specific functions of integrins during metamorphosis will provide a better understanding of how these critical cell surface receptors exert their multiple effects during development (D'Avino, 2000).

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. A temperature-sensitive EcR mutation, EcRA483T, has been identified from a previously isolated collection of EcR mutations. EcRA483T is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages (Carney, 2000).

These observations are consistent with the notion that the defective and degenerating egg chambers observed in EcR mutant females may result from action of EcR and USP heterodimers in ovarian germ cells and/or somatic cells. Recent analysis of expression and function of the ecdysone-dependent E75 early gene during oogenesis has led to the proposal of an ecdysone-dependent checkpoint in midoogenesis. In this study, removal of EcR through creation of EcR germline clones appears to result in egg chamber arrest at stages 6-7, suggesting a requirement for EcR in the germline for progression through oogenesis. Thus, the reproductive defects in EcR mutant females described here are likely to result in part from loss of EcR function in germline cells (Carney, 2000).

Vitellogenesis begins with yolk protein synthesis in the fat body and ovarian follicle cells: ecdysone signaling has been implicated in the control of fat body yolk protein synthesis. It is presumed that active ecdysone receptors are present in the fat body. Because of the detection of ecdysone binding activity in this tissue, the presence of EcR protein in the adult fat body has not been examined directly. In experiments using a yolk protein polyclonal antiserum, small but reproducible decreases are seen in yolk protein production in EcR mutant females compared to controls between 12 and 30 hr following eclosion. It is speculated that the small reductions in yolk protein accumulation seen in EcR mutant females are due to a requirement for EcR in the fat body for yolk protein synthesis. If this is the case, methods that allow a more complete inactivation of EcR functions would be predicted to show more dramatic effects on yolk protein production (Carney, 2000).

EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue

The three Drosophila EcR isoforms differ only at their N termini; thus, they share the conserved ligand-binding domain transcriptional activation function (AF2) and only differ in the unconserved A/B region, which contains a second, isoform-specific, activation function (AF1). A dominant-negative mutant EcR (EcR-DN) has been developed: it was expressed in flies with the GAL4/UAS system, and it was used to block ecdysone signaling in eight tissues or groups of tissues. Localized EcR-DN arrests ecdysone-dependent development in the target cells and often (because of a molting checkpoint) arrests development globally. Simultaneously expressing individual wild-type EcR isoforms in the same target tissues suppresses the EcR-DN phenotype and identifies the rescuing isoform as sufficient to support the development of the target. Every isoform, and even an N-terminal truncated EcR that lacks any AF1, supports development in the fat body, eye discs, salivary glands, EH-secreting neurosecretory cells and in the dpp expression domain, implying that AF1 is dispensable in these tissues. By contrast, only EcR-A is able to support development in the margins of the wing discs, and only EcR-B2 can do so in the larval epidermis and the border cells of the developing egg chamber. In light of these results, the simplest explanations for the widespread spatial and temporal variations in EcR isoform titers appear untenable (Cherbas, 2003).

In comparison with most other transcriptional regulators, the nuclear receptors are remarkably complex. This study is concerned with one aspect of that complexity: the alternative pathways by which a nuclear receptor can activate transcription. A typical nuclear receptor contains two activation surfaces: a strongly conserved activation function (AF2) in its ligand-binding domain (LBD or E region) and a second activation function (AF1) within its unconserved N-terminal A/B region. A great deal is known about the role of AF2 in seeding coactivator complexes, and rather less about AF1. Nonetheless, it is clear from assays in different settings that the two AFs play separable, though often complementary, roles. Thus, depending upon the promoter being tested, its chromatin structure and the host cell, full activation may require either AF or both (Cherbas, 2003 and references therein).

An attractive inference is that these differing AF requirements reflect the diversity of the rate-limiting steps that can be involved in the assembly of functional transcription complexes. If so, it is interesting to consider how these requirements are organized globally. One plausible hypothesis is that AF requirements are simply promoter specific. According to this idea, the set of hormone-responsive promoters in any given cell type may approximate a random sample of AF requirements. A corollary is that, if AF1-requiring promoters are prevalent, every cell type should require AF1, and if the receptor exists in multiple isoforms with different AF1s, every cell should require multiple isoforms (Cherbas, 2003).

A very different hypothesis is suggested by observations on receptors with alternative, isoform-specific AF1s and particularly by the behavior of such receptors in developmental systems. Many nuclear receptors exist as functionally distinct isoforms differing only in their N-terminal domains (e.g. RARs alpha, ß and gamma, RXRs alpha, ß and gamma, TRß, GR, PR, ERalpha and EcR). In addition, receptor isotypes, generated from distinct genes, most often differ in their N-terminal domains (e.g. TR and ER). Where a receptor exists in several isoforms or isotypes, these are generally distributed in a strongly tissue-specific manner. Differences in isoform titers can be quite striking, and for some nuclear receptors there is a strong and unmistakable correlation with developmental fate. Given that alternative AF1s have distinct transcriptional effects and that they can activate distinct sets of target genes, it is reasonable to suppose that the availability of particular AF1s (in specific receptor isoforms or isotypes) may regulate tissue-specific gene expression. Taken to the extreme, this hypothesis would imply that promoters that are induced in all tissues require only AF2, while promoters whose hormone response is tissue-specific require AF1, and in any given cell type the AF1-specific promoters share a requirement for a specific receptor variant (Cherbas, 2003 and references therein).

These alternatives led to the following question: where isoform (hence AF1) titers vary in a tissue-specific way, are they strongly correlated with tissue-specific requirements for the corresponding AF1s? This question has been addressed using mutant animals. For example, the phenotypes of RXRalpha-null mice and mice homozygous for a deletion of the AF1 region of RXRalpha, were compared. Animals that lacked AF1 exhibited localized defects, suggesting specific AF1 requirements, though most RXRalpha functions occurred normally. In a parallel approach, the roles of individual isoforms have been assessed by using isoform-specific mutations. In all these instances, the use of mutant organisms creates daunting interpretive challenges, for it is difficult to decipher the direct, local consequences of receptor failure in the face of numerous organism-wide defects (Cherbas, 2003 and references therein). This study adopts a different approach, arresting receptor function in targeted developmental domains and then testing the abilities of particular receptor isoforms to rescue development when expressed in those domains (Cherbas, 2003).

In Drosophila, there are three EcR isoforms that differ only in their N-terminal regions, the three isoforms being derived from a single structural gene by both alternative promoter usage and alternative splicing. The potencies of the three isoforms have been tested in an EcR-deficient Drosophila cell line, in yeast and in mammalian cells. These studies confirm that the individual isoforms (and their isolated N-terminal regions tested as fusions) differ markedly in their abilities to activate particular test promoters, and that each A/B region contains an AF1 that can activate transcription in an appropriate experimental setting. For example, AF1s from isoforms B1 and B2, but not from A, are active when tested in Drosophila cells with an artificial promoter derived from the Drosophila Eip71CD gene. It is important to note here that these specific isoform requirements are absolute; that is, increasing the titer of an inactive isoform does not increase its ability to activate transcription (Cherbas, 2003 and references therein).

Little is known about the distribution of isoform EcR-B2, but it is clear that isoforms EcR-A and EcR-B1 have very different tissue distributions, and their relative titers in different tissues are well correlated with the fates of those tissues during metamorphosis. For example, immunohistochemistry shows that in third-instar larvae, B1 predominates in larval tissues that will die during metamorphosis, while A predominates in the imaginal discs (Cherbas, 2003 and references therein).

Genetic studies suggest that the isoforms have overlapping but distinct functions during fly development. Of mutations that eliminate all isoforms, only isoform B1, or isoforms B1 and B2 have distinct lethal phases, leading to death at hatching, at pupariation and during larval life, respectively. It is known that ectopic expression of any single EcR isoform can partially rescue development in EcR- animals, with the extent of rescue depending on the isoform (Cherbas, 2003 and references therein).

The GAL4 driver system and dominant-negative mutant EcRs were used in this study to arrest ecdysone receptor function in selected developmental domains. Then, using the same driver system to express individual EcR isoforms, it was asked which isoforms are sufficient to restore and sustain development. Serendipitously, these experiments have revealed a molting checkpoint -- a global block in development induced by local lesions. The developmental arrest associated with that checkpoint is noteworthy in its own right; in the present context it has proven exceptionally useful for these experiments (Cherbas, 2003).

Dominant negative Ecdysone receptor proteins (EcR-DNs) EcR-F645A and EcR-W650A were constructed expecting that, like similar helix 12 mutant TRß proteins, they would lack the transcriptional activation function AF2 and that they might disrupt signaling by competing with wild-type EcR. Both predictions were confirmed by assays in cultured cells, and the experiments presented in this study suggest that EcR-F645A expression can block ecdysone signaling in any Drosophila cell. It is suspected that in vivo EcR-F645A dimerizes with USP and binds to ecdysone response elements, forming unliganded complexes that repress transcription normally, but cannot be converted into activating complexes by ligand (ecdysone). Although still unproven, this scheme conforms to observations in cultured cells and in vitro and is consistent with the vertebrate precedents. This mechanism would account for the interference of EcR-F645A with ecdysone-mediated transcriptional activation. It seems plausible that the mutant EcR also interferes with ecdysone-mediated transcriptional repression, but these experiments did not address this directly (Cherbas, 2003).

The mutant EcRs retain the wild-type B1 N terminus, which has AF1 activity in the wild-type molecule. If AF1 in the mutant EcR mediates transcriptional activation, it would complicate the interpretation of the results. However, this is not the case, for several reasons: (1) co-repressors block AF1 activity; (2) when tested in EcR-deficient cultured cells, under conditions where AF1 is functional in wild-type EcR-B1, EcR-F645A completely fails to activate transcription in response to ecdysone, and (3) in flies, F645A and W650A proteins based on EcR-B2, EcR-A or EcR-C produce results that are qualitatively similar to those described in this study (Cherbas, 2003).

Despite residual uncertainties about the molecular details, it is clear that the EcR-DNs block development in individual tissues much as one would expect if ecdysone action had been arrested. Perhaps most telling is the abrogation of all hormone-induced puffing changes in the salivary gland polytene chromosomes. All the phenotypes observed are associated with a reduction in the level of functional EcR/USP as they are strongly enhanced in a background of lowered wild-type EcR titer and strongly suppressed when extra wild-type EcR is supplied. Analysis in cultured cells shows that these mutant EcRs interfere with wild-type EcR function only when they are present in substantial excess; it is inferred that this is the case in the targeted fly tissues (Cherbas, 2003).

An unanticipated phenotypic consequence of targeted EcR-DN expression is the global block that is referred to as a 'molting checkpoint'. When EcR-DNs are expressed in some tissues, they cause both local phenotypes and a global effect: metamorphosis stops at the time of the next ecdysone-dependent event. This is most clearly illustrated by EcR-DN expression in the Serrate domain (referring to the use of the Serrate promoter to positionally regulate ectopic expression); in this case, expression of the EcR-DN is restricted to the margins of the wing and leg discs, but development is blocked in the entire animal. The term 'molting checkpoint' is used to describe the global block. A similar phenomenon occurs in all other cases where EcR-DN is expressed in epidermal cells. The expression patterns of the Eip, Dpp and GMR drivers are more complex, opening the possibility that the global block results from localized malfunction of crucial tissues such as neurosecretory cells or tracheae; nonetheless, the similarity of the lethal phenotypes makes it attractive to hypothesize that the molting checkpoint is a general consequence of EcR-DN expression in epidermal cells (Cherbas, 2003).

A few basic properties of the molting check-point are important here. (1) Checkpoint arrest is enhanced when EcR levels are reduced and suppressed by rescue constructs; thus, arrest behaves as a downstream consequence of aborted receptor function. (2) The global block to development is invoked by stimuli other than interference with ecdysone signaling. For example, expression of the cell death gene reaper in the GMR domain can produce a similar response. If the molting checkpoint is triggered by some general aspect of defective cells the checkpoint is probably closely related to another phenomenon that has been known for many years, i.e. physical injuries, regeneration or genetic disc-ablation delay in molting. (3) Imaginal discs from arrested animals can be induced to develop by hormone treatment in vitro; it is inferred that metamorphosis stops because the ecdysone titer is insufficient (Cherbas, 2003).

The temporal progression of the cell cycle must be coordinated and this coordination is achieved by a series of checkpoints responding to aberrant events. Similarly, the complex multicellular events of molting and metamorphosis must be coordinated, and it is suggested that the style of this coordination is similar: progress is delayed by a checkpoint invoked by aberrant development. The checkpoint engendered great interest because it revealed itself as a global effect in experiments designed to test purely local phenotypes. It seems likely that the same phenomenon occurs in experiments where targeted expression is not involved; i.e. mutations with diverse, local effects may, because they invoke the checkpoint, reveal themselves by the common phenotype of late larval or pupal lethality (Cherbas, 2003).

The checkpoint is important here because it provides a simple quantitative measure of EcR-DN phenotypes. The frequency of adult eclosion was used as a quantitative indicator of the localized effects of EcR-DN action in several domains. It is the only available assay for the Serrate domain, where the checkpoint acts so efficiently that no escapers were observed under any conditions, and it is a convenient assay in the dpp domain. Its validity is clear in the GMR domain, where the morphological defects in escapers are proportional to the frequency of the block to pupation over a wide range of temperatures and responder insertions. Lethality caused by EcR-DN expression in the Lsp domain arises from a different mechanism; the animals die late in adult development rather than simply failing to molt (Cherbas, 2003).

For those drivers that yield viable, morphologically defective adults, targeted expression of EcR-DNs provides a new way to identify ecdysone-dependent developmental steps. Although none the EcR-DN-induced morphological phenotypes were investigated in detail, the results do support several significant inferences (Cherbas, 2003).

In the GMR, dpp, Lsp2 and EH domains any EcR isoform will support metamorphosis, and, for at least the first three, so will EcR-C. This is even more impressive because two of the domains (GMR and dpp) include diverse cell types. In these four domains, ecdysone-induced transcriptional changes may be mediated by EcR-AF2, by release of EcR/USP-mediated inhibition, or by USP. There is structural evidence to suggest that USP may not be capable of activation and it does not contribute to activation in a cell culture model system, but the first two possibilities are entirely plausible and cannot be distinguished by the experiments described here (Cherbas, 2003).

The contrary result was also observed. In the Ser domain, only EcR-A gives full rescue, and in the Eip and slbo domains only EcR-B2 is effective. Although these effects may be exaggerated by differences in the levels of expression of the responders used, the clear-cut nature of the differences suggest that they are real. If the interpretation of these results is correct, then in each of these domains at least one crucial promoter requires an isoform-specific EcR AF1 (Cherbas, 2003).

Salivary gland puffing might be expected to reveal gene-specific isoform requirements with individual isoforms giving different uncoordinated responses. Instead, the rescued puffing response, at least during its pre-puparial stages, can be characterize by a single parameter -- its rate. Each of the three isoforms supports the normal, coordinate response. It is thought to be simplest to suppose that the AF1s play no role and that the puffing response is simply a sensitive reporter of the expression levels for the three rescuing transgenes. By contrast, earlier studies using heat shock pulses of single isoforms in an EcR-null background, controlled approximately for protein levels, observed the normal pattern rescued by B1>B2>>>A. Plainly, it will require a more sophisticated experiment to determine with confidence whether the isoforms differ in their ability to support the puffing pathway (Cherbas, 2003).

At pupariation, the rescued puffing responses become uncoordinated. This probably reflects the superimposition of the still-in-progress early response with new gene activities induced by the declining ecdysone titer at pupariation (Cherbas, 2003).

The results reported here must be put into the context of previous work on the tissue-specific properties of the EcR isoforms. Using different approaches to local rescue, others have demonstrated that isoforms B1 and B2 (but not A) can support the remodeling of mushroom-body gamma neurons and of the SCP-staining neurons . When EcR mutations are examined at the level of the whole organism, the effects tend to be widespread. Thus, common region EcR nulls are early embryonic lethals, and EcR-B1 nulls are nonpupariating lethals with defects in the leg discs, the imaginal cells of the midgut islands, the larval gut and the histoblasts. Animals null for both EcR-B1 and -B2 are early larval lethals. The widespread nature of the defects in these mutant animals makes it impossible to judge the localized requirements for EcR isoforms (Cherbas, 2003).

In addition to testing the capacity of individual isoforms to support development in diverse tissues, these experiments contribute to the catalog of isoform distribution in those tissues. The distribution of isoforms is complex both in time and space. Previous experiments used immunostaining to examine the relative levels of isoforms A and B1 in late third-instar tissues and in the CNS. The measurements of the effects of isoform-specific mutations on EcR-DN phenotypes contribute an estimate of the contribution of individual endogenous isoforms in the particular times and places in which each driver is expressed. These data imply major roles for isoform B2 in the larval fat body and epidermis. In addition, only isoform B2 can rescue the EcR-DN effects in the slbo domain, suggesting that it may also be the major isoform in the follicle cells (Cherbas, 2003).

Two alternative models have been proposed for the distribution of AF1-specific promoters. According to one, individual domains should exhibit specific isoform requirements that can be predicted by their isoform contents. The results lend no support to this idea. It is not known why isoform titers vary, but they do not appear to be good predictors of isoform requirements. Instead, the results are consistent with the following picture: A small minority of promoters require specific isoforms. As each responding tissue may contain several (or many) critical promoters, specific AF1 requirements are not limited to a small minority of tissues. Still, it is remarkable that tissues lacking even one such critical promoter are not rare. In those tissues (about half of the sample) AF1 is dispensable, and ecdysone effects are mediated by AF2 or by relief of repression. In some cells, at least one critical promoter does require a specific AF1. When many tissues lack a specific isoform, developmental defects are likely and their phenotypes depend on both the missing isoform and the intervention of the molting checkpoint (Cherbas, 2003).

Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals, results supporting this hypothesis were obtained. Tests were performed for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, it was found that both EcR-B isoforms are able to rescue the neuronal defect cell autonomously, but EcR-A is less effective. The effect of over-expressing the isoforms was examined in a wild-type background. A sensitive period was determined when high levels of either EcR-B isoform are lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast has no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppress puparial tanning, and result in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect (Schubiger, 2003).

Loss of function of both EcR-B isoforms inhibits dendritic pruning by the larval Tv-neurons, a set of thoracic FMRFamide expressing neurons. However, since all cells in these animals are mutant, it was not known if the failure to prune dendrites was a cell autonomous defect. Consequently, it was asked if the dendritic pruning defect could be rescued by expressing the different EcR isoforms specifically in these neurons. Using the FG5-Gal4 driver containing regulatory sequences of the FMRFamide gene wild-type EcR isoforms as well as the GFP-labeled membrane marker mCD8 were expressed in EcR-B mutant animals. The cell-autonomous rescue of pruning in the Tv neurons shows that given the correct set of receptors in these neurons, ecdysone can induce a pruning response even in a mutant environment in which overall nervous system development is arrested. Both the pruning of these neurosecretory cells and the pruning of the mushroom body neurons require the presence of an EcR-B isoform. The pruning of the Tv-cells, however, is only inhibited when both EcR-B1 and EcR-B2 are absent. This differs from the mushroom body neurons that fail to undergo pruning when only EcR-B1 is missing. Nevertheless similarity across different neuronal types suggests that a general set of rules may be employed early in metamorphosis to remove larval specializations (Schubiger, 2003).

Cellular mechanisms of dendrite pruning in Drosophila; The expression of dominant negative EcR in neurons that are fated to die blocks their death and prevents local degeneration

Regressive events that refine exuberant or inaccurate connections are critical in neuronal development. Multi-photon, time-lapse imaging was used to examine how dendrites of Drosophila dendritic arborizing (da) sensory neurons are eliminated during early metamorphosis, and how intrinsic and extrinsic cellular mechanisms control this deconstruction. Removal of the larval dendritic arbor involves two mechanisms: local degeneration and branch retraction. In local degeneration, major branch severing events entail focal disruption of the microtubule cytoskeleton, followed by thinning of the disrupted region, severing and fragmentation. Retraction was observed at distal tips of branches and in proximal stumps after severing events. The pruning program of da neuron dendrites is steroid induced; cell-autonomous dominant-negative inhibition of steroid action blocks local degeneration, although retraction events still occur. The data suggest that steroid-induced changes in the epidermis may contribute to dendritic retraction. Finally, it was found that phagocytic blood cells not only engulf neuronal debris but also attack and sever intact branches that show signs of destabilization (Williams, 2005).

The abdominal sensory system of the adult fly is a mosaic consisting of postembryonic neurons derived from the imaginal histoblast nests and genital disc, and a subset of larval sensory neurons of embryonic origin. At the onset of metamorphosis, larval sensory neurons either die or survive and remodel. Among the dorsal neurons of abdominal segments 2-5, it was found that dorsal dendritic arborizing ddaA, ddaF and ddaB die whilst ddaC, ddaD and ddaE survive (Williams, 2005).

Programmed cell death is a near ubiquitous phenomenon in the vertebrate nervous system and essential for proper morphogenesis. Surprisingly, most studies have focused on the cell body and paid little attention to the fate of neuritic processes, even though the axons of most projection neurons have reached their targets before the onset of cell death. Time-lapse imaging was used to observe the dendrites of dying neurons in intact animals. The distal branches of the ddaF develop swellings at 2 h APF that become distinct beads, which accumulate tubulin. Between 3 and 4 h APF branches undergo severing at multiple sites where they have thinned. This beading propagates through the arbor in a proximal to distal direction with proximal changes occurring 20-30 minutes ahead of the distal ones. This same progression is seen in ddaB but with a lag of 1 hour. A similar degeneration is found in the peripheral arbors of Rohon-Beard neurons, which also thin and fragment when dying (Williams, 2005).

The destruction of peripheral processes has been most widely studied following acute local trauma, where the axon branch distal to the lesion undergoes a stereotyped degeneration by fragmentation, termed Wallerian degeneration (Waller, 1850). There are similarities between the degeneration of dendrites in the current study and the axon pathology seen in Wallerian degeneration. (1) In vitro models of Wallerian degeneration reveal a proximal to distal spread of beading in the separated distal neurites. (2) The microtubule cytoskeleton is disrupted following transection and the beaded morphology and redistribution of tubulin seen in the da neuron dendrites resembles that seen in neurons treated with microtubule depolymerizing drugs (Williams, 2005).

The expression of EcRDN in neurons that are fated to die blocks their death and prevents the local degeneration seen between 3 and 6 h APF. This prompted an inquiry as to whether the degeneration normally observed is due to cell death directly or is the result of a hormone-induced microtubule disassembly program running in parallel with cell death. When cell death was blocked by expressing p35, the dendrites showed no signs of degeneration by 5 h APF, yet by 18 h APF their arbor was largely removed. Thus, with p35, the dendrites of these doomed cells prune at the same time and in the same way as ddaD and ddaE, suggesting that under normal conditions, death directly causes the dendrite degeneration observed in ddaA, ddaF and ddaB at 3-6 h APF. Similar observations made after killing da neurons by laser ablation support this proposition (Williams, 2005).

Pruning of the dendrites of ddaD and ddaE starts between 6 and 10 h APF and is largely complete by 24 h APF. Time-lapse movies reveal that dendrites undergo deconstruction by two different cellular mechanisms: local degeneration and branch retraction (Williams, 2005).

During local degeneration, branches are detached from the main arbor and undergo fragmentation. Axons and dendrites of mushroom body neurons undergo local degeneration: between 4 and 6 h APF the processes undergo blebbing, and by 8 h APF most of the dendritic arbor has been removed. By 18 h APF the axons have fragmented, and during that time there is no evidence to suggest that gamma neurons undergo retraction. Similarly, larval projection neurons in the olfactory lobe undergo local degeneration (Williams, 2005).

A key step in local degeneration appears to be severing, which it was possible to visualize with time-lapse movies. Severing happens in one of two ways, depending on position within the arbor. In proximal regions, severing is preceded by thinning. Once a branch has thinned, it severs and the stump retracts, while the separated arbor undergoes fragmentation. It was found that beads adjacent to thinning regions contain abundant Futsch and tub::GFP, whereas these are lacking in the thinned regions, suggesting that the bulk of the microtubule cytoskeleton is lost in these focal regions. This supports observations that myc-marked tubulin disappears from pruning gamma neurons before the axon is lost. Similarly, it has been shown that just prior to shedding 'axosomes', the proximal region of a retracting axon becomes devoid of organized microtubules. When neurons express EcRDN, the caliber of the proximal branches does not change, suggesting that a redistribution of the microtubule cytoskeleton fails and proximal severing is subsequently blocked (Williams, 2005).

The second type of severing occurs at more distal sites within the arbor. Here there is no thinning and only occasional beading; thus it is possible that different mechanisms are responsible for severing at distal sites. Nevertheless, distal severing is also suppressed in neurons that express EcRDN, suggesting that a destabilization is also important here (Williams, 2005).

After severing, both proximal and distally detached branches undergo fragmentation. The branches thin while beads form along their length and break at multiple sites, generating GFP blebs that are removed by phagocytes. Although most of the events observed in branch fragmentation during pruning appear similar to those seen in the arbors of dying da neurons, an exception is the appearance of filopodia. When the microtubule cytoskeleton and the membrane were simultaneously imaged in pruning neurons, it was often found that filopodia are coincident with the areas of microtubule disorganization. Lateral filopodia have been found when colcemid was applied to chick neurons in culture. Filopodia do not appear on the arbors of the doomed cells since the cells are undergoing programmed cell death. However, when cell death is blocked by expression of p35 these cells later show abundant filopodia while their dendrites are removed. This suggests a fundamental difference between the dendrite removal observed during the pruning of ddaD and ddaE, and that seen in cells that undergo programmed cell death (Williams, 2005).

The primary branches of ddaD and ddaE can be retracted. These retraction events occurred in 6 of 17 neurons imaged and the branches of such neurons often possessed many filopodia. When retraction events happen, local degeneration may still occur in secondary branches and on other primary branches nearby. It is somewhat problematic to apportion what percentage of pruning is due to retraction and what is due to severing and fragmentation, since they both appear to happen simultaneously, e.g. where a branch is in the process of distal retraction and suddenly is severed at a more proximal site (Williams, 2005).

The dendrites of neurons expressing EcRDN form bulb-like structures on their distal tips and produce few if any filopodia. Local degeneration is blocked in these neurons, but they do eventually remove their dendrites by retraction. A number of scenarios could explain this retraction phenotype. It is possible that EcRDN does not block all ecdysone signaling and so the cell intrinsic ecdysone pruning program has not been entirely eliminated. It is also possible, but unlikely, that the EcRDN results in a non-specific 'neomorphic' since the specificity of EcRDN has been demonstrated. The other possibility is that EcRDN completely blocks ecdysone signaling and that another parallel intrinsic signaling pathway plays a role in dendrite retraction. It is most likely that EcRDN completely blocks ecdysone signaling and that the phenotype that is seen reveals extrinsic factors that are important for dendrite pruning under wild-type conditions. The neuron-specific expression of EcRDN means that ecdysone signaling is disrupted only in the neuron and that the local environment can undergo its normal hormone induced changes (Williams, 2005).

A dorsal region of the epidermis is the target for the dendrites of ddaD and ddaE. Two potential interactions could be taking place between the target and pruning arbor. The epidermis could be sending an instructive signal causing the arbor to prune or there could be a passive loss of epidermal contacts as the larval epidermis is replaced. To explore the latter possibility, movies were made at the time when ddaD and ddaE are pruning. It has been widely held that the larval epidermal cells are removed only by the migrating front of adult epidermal cells derived from the histoblast nest. Surprisingly it was found that during early metamorphosis larval cells are removed at sites distant from the histoblast migration front. The epidermal cells are removed by phagocytic blood cells and the cells that are removed do not show obvious signs of apoptosis before being contacted by the phagocyte. This suggests that the larval cells are killed by a phagocytosis-induced cell death as described in C. elegans and Drosophila. Importantly, when a larval cell is removed, other cells move to compensate for its loss. Thus the epidermis becomes a dynamic substrate during prepupal and early pupal development, this movement may result in shearing events in distal dendritic branches and could explain the phenomenon of distal severing without thinning. Likewise, the disruption of neuron-epidermal contacts may contribute to the retraction of primary branches seen in an extreme form when neurons express the EcRDN (Williams, 2005).

Drosophila dSet2 functions in H3-K36 methylation and and genetically interacts with EcR

Drosophila Set2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of Set2 lack Set2 expression and H3-K36 methylation, indicating that Set2 is the sole enzyme responsible for this modification in Drosophila. Set2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdysone-controlled genetic program. A transheterozygous EcR null mutation/Set2 RNAi combination produces a complete (balloon) separation of the wing surfaces, revealing a genetic interaction between the Ecdysone receptor (EcR) and Set2. Immunoprecipitation studies demonstrated that Set2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII) (Stabell, 2007).

The expression level of Set2 during development was determined by RT-PCR. Set2 is expressed throughout the life cycle of Drosophila. Whereas the level of expression is rather low in 0-6 h embryos it increases to an apparently steady level after that time-point, with a noticeable peak in late third instar larvae. It is interesting to note that the major expression of Set2 in adults is in the gonads (Stabell, 2007).

To investigate the function of Set2, RNA interference (RNAi) was used to lower Set2 mRNA and protein levels. Fly stocks were generated carrying the responder transposon UAS-dSet2.IR, in which a Gal4-dependent promoter expresses the inverted repeat of Set2 to yield a double-stranded RNA molecule. The pUdsGFP vector used for this purpose has an independent UAS-GFP marker so that a tissue exposed to RNAi will simultaneously show GFP expression. Different driver stocks, each expressing Gal4 in a temporally and spatially distinct developmental domain, were selected, and the stocks were crossed to yield hybrid progeny with targeted UAS-dSet2.IR expression (Stabell, 2007).

Initially, a salivary gland-specific driver, Sgs3-Gal4 was used. This allowed immunostaining to be used against Set2 and H3-K36Me, and at the same time visualization of nuclei with DAPI staining and GFP fluorescence. Immunostaining of wild type salivary glands with anti-Set2 and anti-H3K36Me, respectively, showed that both Set2 and dimethylated of H3-K36 are present in the cells. The effect of RNAi knockdown of Set2 by the Sgs3-Gal4 driver was analyzed by immunostaining. Salivary glands from UAS-dSet2/Sgs3-Gal4 larvae were dissected at 3 ± 1 h before puparium formation and immunostained with anti-Set2, anti-dimethyl H3-K4, anti-dimethyl H3-K27, anti-dimethyl H3-K36, anti-H3-9Me, and anti-acetyl H3-K9. Immunostaining with anti-Set2 reveals the absence of Set2 protein in salivary gland cells where UAS-dSet2.IR is expressed by the Sgs3-Gal4 driver. During this work two nuclei in one salivary gland lobe (out of 10 pairs) were found that did not express GFP but were clearly detectable by DAPI staining. Immunostaining with anti-H3-K36Me reveals the absence of H3-K36 dimethylation in salivary gland cells where UAS-dSet2.IR is expressed. Most importantly, however, UAS-dSet2.IR is not induced in the two salivary gland cells not expressing GFP, and here the H3-K36 dimethylation is clearly observable. Furthermore, fat body cells, where the Sgs3-Gal4 driver is not expressed, also show H3-K36 dimethylation in all nuclei. The reason to why the two cells have lost the ability to express the GFP-tagged UAS-dSet2.IR construct may be either loss of UAS or Gal4 sequences or the whole construct(s) prior to the onset of polyploidization. Methylation and acetylation of H3-K9, respectively, are unaffected by the absence of Set2. This also is true for H3-K4 and H3-K27, thus showing the specificity of Set2 RNAi. It is concluded that the Set2 gene encodes a HKMT responsible for all detectable H3-K36 dimethylation in Drosophila (Stabell, 2007).

Set2 interacts genetically with the ecdysone receptor: To test whether a knock down of Set2 affects the fly, the ubiquitously expressed da-Gal4 driver was crossed with UAS-dSet2.IR flies. All progeny are da-Gal4/UAS-dSet2.IR and develop normally until the end of the third larval instar. However, these RNAi larvae do not form their puparium and crawling larvae are found after 10-12 days. The larvae finally stop moving, and in a few cases melanin-less 'pseudo-prepupae' are formed, which maintain the elongated larval form and fail to evert the anterior spiracles. These observations may indicate a defect in ecdysone responses at puparium formation, similar to those reported for mutants in the ecdysone pathways, and thus implicates Set2 in this regulatory cascade (Stabell, 2007).

The defects in puparium formation seen in Set2 RNAi animals could result from either a decrease in the ecdysone titer or a decrease in the ability of the ecdysone signal to be transduced. To distinguish between these possibilities the effects were examined of feeding ecdysone to Set2 RNAi larvae. This method has been shown to effectively rescue phenotypes associated with ecdysone-deficient mutations. Mid- and late-third instar larvae were transferred to food either with or without 20-hydroxyecdysone (20E) for 6-8 h and scored on a 12 h basis. Feeding 20E to Set2 RNAi larvae did not rescue them to puparium formation. Therefore, it is concluded that ecdysone is not limiting in the da-Gal4/UAS-dSet2.IR animals and that Set2 functions downstream of ecdysone biosynthesis and release (Stabell, 2007).

The development of a flat bilayered wing from an imaginal disc monolayer involves four steps that occur twice during metamorphosis. The timing of these two rounds of wing morphogenesis correlates with the two main ecdysone peaks during metamorphosis; namely during mid- and late prepupal stages, and from 24 to 72 h after puparium formation, and it has been shown that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. The effects of EcR mutations on integrin transcription and the essential role for integrins during wing morphogenesis are both principally restricted to the pupal stages. To investigate if Set2 might be involved in wing morphogenesis, Set2 RNAi was triggered in the wing discs by the ap-Gal4 driver. Induction of Set2 RNAi by the ap-Gal4 driver generates a blister in the proximal (towards thorax) part of the wings. The transparent blisters are initially large balloons filled with air and hemolymph that deflate after a while, leaving a scar that may cause the wing to bend up. This phenotype shows 100% penetrance (Stabell, 2007).

Ecdysone exerts its effects on development through a heterodimer of two nuclear receptors, encoded by EcR and usp. The ecdysone/EcR/USP complex then directly activates cascades of gene expression. To assess if Set2 and EcR genetically interacts in a common pathway controlling wing morphogenesis, whether the EcR null mutation EcRM554fs could dominantly affect the blistered wing phenotype of ap-Gal4/+;UAS-dSet2.IR/+ flies described above was tested. Therefore, the stock y w;EcRM554fs/CyO;UAS-dSet2.IR was synthesized and crossed reciprocally to y w;ap-Gal4/CyO flies. EcRM554fs dramatically enhances the blistered wing phenotype in all EcRM554fs/ap-Gal4;UAS-dSet2.IR/+ flies. The wing layers were completely separated (ballooned) and often one or both wings were held in a Dichaete-like fashion. The balloon soon becomes blackened and remains inflated for 1-2 days. Again, all flies of the genotype ap-Gal4/CyO;UAS-dSet2.IR/+ have the blistered wing phenotype. It should also be mentioned that heterozygous EcRM554fs/CyO;UAS-dSet2.IR/+ flies have normal wings. Taken together, these results suggest that Set2 and EcR may function in a common ecdysone signaling pathway during wing morphogenesis, and that this interaction may be dose sensitive (Stabell, 2007).

These results prompted an analysis by RT-PCR of the ecdysone receptor genes DHR3, DHR4 and reaper (rpr) in dSet2.IR animals around pupariation. While an apparently normal expression pattern for DHR3, DHR4 and rpr was observed at the same time points in control salivary glands, these genes show a clear decline in mRNA levels in Set2 RNAi salivary glands. Both DHR3 and DHR4 execute essential functions during development, including at the onset of metamorphosis. The parallel functions of these two nuclear receptors suggest that they act together to direct the switch from late-larval to prepupal genetic programmes. Although these results were obtained from Set2 RNAi in salivary glands it is believed that these genes, and probably several others, also are affected in da-Gal4/UAS-dSet2.IR larvae, thus explaining the failure of these larvae to pupariate. The fact that none of the studied genes is fully down regulated also suggests that the non-pupariating phenotype may be ascribed to a collective down regulation of essential genes involved in the larval-to-pupal transition. It is also interesting to note the decreased level of rpr mRNA, which is significant at +2 h. Although rpr is not involved in metamorphosis other than in programmed cell death, its down regulation supports the notion that Set2 is linked to the ecdysone regulatory hierarchy (Stabell, 2007).

Recent reports have shown that Set2 from various organisms binds to the hyperphosphorylated CTD of RNAPII, implying that K36 methylation plays an important role in the transcription elongation process. The presence of both the WW and SRI domains suggested that Set2 may associate with RNAPII also in Drosophila. To address this issue, extracts prepared from control or Set2 RNAi embryos were immunoprecipitated with antibodies directed against Ser5-phosphorylated CTD, followed by immunoblotting with antibodies directed against Set2 and Ser5-phosphorylated CTD form of RNAPII. Immunoprecipitation of Ser5-phosphorylated CTD resulted in strong immunoreactivity of both phosphorylated CTD and Set2 in control embryos whereas no Set2 is detected in extracts from RNAi embryos. This result was corroborated by showing co-localization of Set2 and elongating RNAPII on salivary gland chromosomes. While these results demonstrate that Set2 is associated with the elongating form of RNAPII in Drosophila, the precise role of this association is currently unclear. However, the fact that a loss of Set2/K36 methylation results in mutant phenotypes associated with defects in the ecdysone response indicates that Set2/K36 methylation plays an important role in the ecdysone regulatory hierarchy (Stabell, 2007).

Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons

Specificity of sensory neurons requires restricted expression of one sensory receptor gene and the exclusion of all others within a given cell. In the Drosophila retina, functional identity of photoreceptors depends on light-sensitive Rhodopsins (Rhs). The much simpler larval eye (Bolwig organ; see The Extraretinal Eyelet of Drosophila: Development, Ultrastructure, and Putative Circadian Function) is composed of about 12 photoreceptors, eight of which are green-sensitive (Rh6) and four blue-sensitive (Rh5). The larval eye becomes the adult extraretinal 'eyelet' composed of four green-sensitive (Rh6) photoreceptors. This study shows that, during metamorphosis, all Rh6 photoreceptors die, whereas the Rh5 photoreceptors switch fate by turning off Rh5 and then turning on Rh6 expression. This switch occurs without apparent changes in the programme of transcription factors that specify larval photoreceptor subtypes. It was also shown that the transcription factor Senseless (Sens) mediates the very different cellular behaviours of Rh5 and Rh6 photoreceptors. Sens is restricted to Rh5 photoreceptors and must be excluded from Rh6 photoreceptors to allow them to die at metamorphosis. Finally, Ecdysone receptor (EcR) was shown to function autonomously both for the death of larval Rh6 photoreceptors and for the sensory switch of Rh5 photoreceptors to express Rh6. This fate switch of functioning, terminally differentiated neurons provides a novel, unexpected example of hard-wired sensory plasticity (Sprecher, 2008).

The adult Drosophila eyelet comprises approximately four photoreceptors located between the retina and the optic ganglia. It directly contacts the pacemaker neurons of the adult fly, the lateral neurons. In conjunction with the compound eye and the clock-neuron intrinsic blue-sensitive receptor cryptochrome it helps shift the phase of the molecular clock in response to light. All eyelet photoreceptors express green-sensitive Rh6, and are derived from photoreceptors of the larval eye that mediate light avoidance and entrainment of the molecular clock by innervating the larval lateral neurons (Sprecher, 2008).

Larval photoreceptors develop in a two-step process during embryogenesis. Primary precursors are specified first and develop as the four Rh5-subtype photoreceptors. They signal through Epidermal growth factor receptor (EGFR) to the surrounding tissue to develop as secondary precursors, which develop into the eight Rh6-subtype photoreceptors. Two transcription factors specify larval photoreceptor subtypes. Spalt (Sal) is exclusively expressed in Rh5 photoreceptors, where it is required for Rh5 expression. Seven-up (Svp) is restricted to Rh6 photoreceptors, where it represses sal and promotes Rh6 expression. A third transcription factor, Orthodenticle (Otd), expressed in all larval photoreceptors, acts only in the Rh5 subtype to promote Rh5 expression and to repress Rh6 (Sprecher, 2008 and references therein).

To address the relation between the larval Rh5 and Rh6 photoreceptors and the adult eyelet, they were tracked through metamorphosis. To permanently label them, UAS-Histone2B::YFP, which is stably incorporated in the chromatin, and thus remains detectable in post-mitotic neurons throughout pupation, was used. Surprisingly, all Rh6 photoreceptors degenerate and disappear during early phases of metamorphosis. In contrast, Rh5 photoreceptors can be followed throughout pupation. Expression of Rh5 ceases during early stages of pupation and, at mid-pupation, neither Rh5 nor Rh6 can be detected. About four cells are still present, however, and can be identified by rh5-Gal4/UAS-H2B::YFP or GMR-Gal4/UAS-H2B::YFP. Eyelet photoreceptors only express Rh6, even though H2B::YFP driven by rh5-Gal4 is detectable in those cells. Therefore, the four larval Rh5 photoreceptors must switch rhodopsin expression at metamorphosis to give rise to the four eyelet Rh6 photoreceptors. The remaining eight Rh6 photoreceptors die, their axon becoming fragmented before disappearing. A 'memory experiment' (rh5-Gal4/UAS-Flp;Act-FRT > STOP > FRT-nlacZ) also showed that eyelet Rh6 photoreceptors did express Rh5 earlier (Sprecher, 2008).

The death of Rh6 photoreceptors and transformation of Rh5 photoreceptors was further verified by three independent sets of experiments (Sprecher, 2008).

(1) Rh5 photoreceptors were ablated by expressing pro-apoptotic genes rpr and hid (rh5-Gal4/UAS-rpr,UAS-hid). This results in the absence of larval Rh5 photoreceptors and the complete absence of the eyelet. Conversely, preventing cell death of the Rh6 subtype by expressing the apoptosis inhibitor p35 (rh6-Gal4/UAS-p35) leads to an eyelet that consists of 12 photoreceptors, all expressing Rh6 (Sprecher, 2008).

(2) Larval Rh6 photoreceptors development was blocked by expressing a dominant negative form of EGFR (so-Gal4/UAS-H2B::YFP; UAS-EGFRDN). The eyelet of these animals is not affected and three or four cells express Rh6 normally. This shows that larval Rh6 photoreceptors do not contribute to the eyelet (Sprecher, 2008).

(3) The expression of Sal (Rh5-subtype specific) and Svp (Rh6-subtype specific) was analyzed in the adult eyelet: eyelet photoreceptors still express Sal, but not Svp even though these photoreceptors now express Rh6. Rh5 requires Sal expression in the Bolwig organ, but Otd function is also necessary to activate Rh5 and to repress Rh6. In otd mutants, larval Rh5 photoreceptors marked by Sal express Rh6 and lack Rh5 expression, thus mimicking the switch at metamorphosis. Thus, Rh6 could be expressed in Rh5 photoreceptors if otd function were lost in the eyelet. However, Otd expression does not change during the transition from the Bolwig organ to eyelet although it might be inactive in the eyelet (Sprecher, 2008).

What is the trigger that controls the switch from rh5 to rh6? Ecdysone controls many developmental processes during metamorphosis. EcR is expressed during the third larval instar and pupation in all larval photoreceptors and surrounding tissues. To evaluate EcR activity, a reporter line was used in which lacZ is under the control of multimerized ecdysone response elements (7XEcRE-lacZ). The expression of lacZ is absent until late third instar and prepupation, whereas thereafter all larval photoreceptors (and surrounding tissue) express 7XEcRE-lacZ. EcR expression decreases during late pupation and is no longer detectable by the time Rh6 expression starts in the eyelet (Sprecher, 2008).

To test the role of ecdysone, a dominant negative form of EcR was expressed specifically in larval Rh5 photoreceptors, while permanently labelling these cells (rh5-Gal4/UAS-H2B::YFP;UAS-EcRDN). This causes no disruption of larval photoreceptor fate, but the eyelet of these animals now consists of four photoreceptors that all express Rh5 instead of Rh6. A comparable phenotype is observed after expression of an RNA interference (RNAi) construct for EcR (rh5-Gal4/UAS-H2B::YFP;UAS-EcRRNAi). Therefore, loss of EcR function prevents larval photoreceptors from switching to Rh6 expression. In both cases, larval Rh6 photoreceptors still degenerate and are not observed in the eyelet (Sprecher, 2008).

The dominant negative form of EcR was also expressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP; UAS-EcRDN). In this case, the Bolwig organ is not affected but the resulting adult eyelet consists of about 12 photoreceptors, all expressing Rh6. This presumably results from Rh6 photoreceptors not undergoing apoptosis whereas larval Rh5 photoreceptors still switch expression to Rh6 in the eyelet. Expression of UAS-EcR-RNAi in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-EcRRNAi) leads to the same results (Sprecher, 2008).

Although EcR could directly control the switch of rhodopsin expression through binding to the promoters of rh5 and rh6, these promoters contain no potential EcR binding sites. Moreover, as no EcR expression is detectable when Rh6 starts to be expressed, this would make it unlikely for EcR to control directly the switch to Rh6. Finally, only allowing expression of the dominant negative form of EcR starting at mid-pupation (GMR-Gal4/Tub-Gal80ts,UAS-EcRDN), after rh5 is switched off, does not prevent activation of Rh6 in the eyelet. Thus EcR most likely acts in an indirect manner in regulating rhodopsins, likely through the activation of transcription factors that bind to rh5 and rh6 promoters (Sprecher, 2008).

The differential response to ecdysone of Rh6 photoreceptors (which die) and of Rh5 photoreceptors (which switch to Rh6) must be due to intrinsic differences between the two subtypes before EcR signalling. Likely candidates are Sal and Svp. However, late misexpression of Svp in Rh5 photoreceptors (rh5-Gal4/UAS-H2B::YFP;UAS-svp) or of Sal in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sal) neither affects rhodopsin expression or cell number in the eyelet nor alter the expression of rhodopsins in the Bolwig organ (which is only affected by very early expression of these transcription factors, through so-Gal4. Thus neither Sal nor Svp are sufficient to alter the response of larval photoreceptors to EcR (Sprecher, 2008).

An additional factor, independent from svp and sal, must therefore allow survival of Rh5 photoreceptors, or promote Rh6 photoreceptor death. It was found that the transcription factor Sens is specifically expressed in larval Rh5 photoreceptors and remains expressed in all cells in the eyelet where it might act to promote cell survival. To test this, sens was misexpressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sens). This results in an eyelet that consists of 12 photoreceptors, all expressing Rh6. Thus, expression of Sens in Rh6 photoreceptors is sufficient to rescue them from death, without affecting Sal and Svp expression and subtype specification of larval photoreceptors (Sprecher, 2008).

Ecdysone hormonal signalling thus acts in two independent ways during the formation of the adult eyelet. First, it induces the degeneration of the Rh6 subtype, thereby assuring the correct number of eyelet photoreceptors. This apoptotic death requires the absence of Sens, whose expression is restricted to Rh5 photoreceptors that survive. Second, ecdysone signalling is also required to trigger the switch of spectral sensitivity of blue-sensitive (Rh5) larval photoreceptors to green-sensitive (Rh6) eyelet photoreceptors (Sprecher, 2008).

Thus terminally differentiated sensory neurons switch specificity by turning off one Rhodopsin and replacing it with another. Although examples of such switches in sensory specificity of terminally differentiated, functional, sensory receptors are extremely rare, this strategy might be more common than currently anticipated. In the Pacific pink salmon and rainbow trout, newly hatched fish express an ultraviolet opsin that changes to a blue opsin as the fish ages. As in flies, this switch might reflect an adaptation of vision to the changing lifestyle. The maturing salmon, born in shallow water, later migrates deeper in the ocean where ultraviolet does not penetrate. The rhodopsin switch in the eyelet may similarly be an adaptation to the deeper location of the eyelet within the head, as light with longer wavelengths (detected by Rh6) penetrates deeper into tissue than light with shorter wavelengths (detected by Rh5) (Sprecher, 2008).

The eyelet functions with retinal photoreceptors and Cryptochrome to entrain the molecular clock in response to light. The larval eye, on the other hand, functions in two distinct processes: for the entrainment of the clock and for the larva to avoid light. Interestingly, the Rh5 subtype appears to support both functions whereas Rh6 photoreceptors only contribute to clock entrainment. Thus, the photoreceptor subtype that supports both functions of the larval eye is the one that is maintained into the adult and becomes the eyelet. Why are Rh6-sensitive photoreceptors not maintained? As these photoreceptors are recruited to the larval eye secondarily, the ancestral Bolwig organ might have had only Rh5 photoreceptors and had to undergo a switch in specificity. Larval Rh5 photoreceptors appear to maintain their overall connectivity to the central pacemaker neurons. However, they are also profoundly restructured and exhibit widely increased connectivity during metamorphosis. This might be due to the increase in number of their target neurons, and the switch of Rh might be part of more extensive plasticity during formation of the eyelet, including increased connectivity and possibly the innervation of novel target neurons (Sprecher, 2008).

The general model that sensory neurons express only a single sensory receptor gene does not hold true for salmon and the fruitfly. Interestingly, reports from several other species, including amphibians, rodents and humans, show co-expression of opsins. In humans, for instance, it has been proposed that cones first express S opsin and later switch to L/M opsin. However, this likely reflects a developmental process rather than a functional adaptation (Sprecher, 2008).

This study identified two major players in the genetic programme for the transformation of the larval eye to the eyelet. (1) EcR acts as a trigger for both rhodopsin switch and apoptosis. Surprisingly, the upstream regulators specifying larval photoreceptor-subtype identity, Sal, Svp and Otd, do not contribute to the genetic programme of sensory plasticity of the rhodopsin switch. Therefore a novel genetic programme is required for regulating rhodopsin expression in the eyelet, which likely depends on downstream effectors of EcR (Sprecher, 2008).

(2) Larval Rh5 and Rh6 photoreceptors respond differently to ecdysone, either switching rhodopsin expression or undergoing apoptosis. This appears to depend on Sens, which is likely to be required for the survival of Rh5 photoreceptors. The role of Sens in inhibiting apoptosis is not unique to this situation: Sens is essential to promote survival of salivary-gland precursors during embryogenesis. The vertebrate homologue of sens, Gfi-1, acts to inhibit apoptosis of T-cell precursors in haematopoiesis and cochlear hair cells of the inner ear. Thus the anti-apoptotic function of Sens/Gfi-1 may be a general property of this molecule (Sprecher, 2008).

Ecdysone acts in remodelling neurons during metamorphosis. In γ-neurons of the mushroom body, a structure involved in learning and memory, ecdysone is required for the pruning of larval processes. Similarly, dendrites of C4da sensory neurons undergo large-scale remodelling that depends on ecdysone signalling. Interestingly, in the moth Manduca, 'lateral neurosecretory cells' express cardio-acceleratory peptide 2, which is switched off in response to ecdysone before expression of the neuropeptide bursicon is initiated in the adult (Sprecher, 2008).

The transformation of larval blue-sensitive photoreceptors to green-sensitive photoreceptors of the eyelet reveals an unexpected example of sensory plasticity by switching rhodopsin gene expression in functional, terminally differentiated sensory neurons (Sprecher, 2008).

Ecdysone signaling regulates the formation of long-term courtship memory in adult Drosophila melanogaster

Improved survival is likely linked to the ability to generate stable memories of significant experiences. Considerable evidence in humans and mammalian model animals shows that steroid hormones, which are released in response to emotionally arousing experiences, have an important role in the consolidation of memories of such events. In insects, ecdysone is the major steroid hormone, and it is well characterized with respect to its essential role in coordinating developmental transitions such as larval molting and metamorphosis. However, the functions of ecdysone in adult physiology remain largely elusive. This study shows that 20-hydroxyecdysone (20E), the active metabolite of ecdysone that is induced by environmental stimuli in adult Drosophila, has an important role in the formation of long-term memory (LTM). In male flies, the levels of 20E were found to be significantly increased after courtship conditioning, and exogenous administration of 20E either enhanced or suppressed courtship LTM, depending on the timing of its administration. Mutants in which ecdysone signaling is reduced are defective in LTM, and an elevation of 20E levels is associated with activation of the cAMP response element binding protein (CREB), an essential regulator of LTM formation. These results demonstrate that the molting steroid hormone ecdysone in adult Drosophila is critical to the evolutionarily conserved strategy that is used for the formation of stable memories. It is proposed that ecdysone is able to consolidate memories possibly by recapturing molecular and cellular processes that are used for normal neural development (Ishimoto, 2009).

The objective of this study was to investigate whether the steroid molting hormone 20E regulates LTM formation in adult Drosophila. This study shows the following; (1) training for courtship-memory leads to an elevation of 20E levels in adult flies; (2) administering exogenous 20E has either a positive or negative effect on courtship LTM, depending on the context; (3) disrupting either ecdysone synthesis or function of the nuclear EcR results in defective LTM; (4) functional ecdysone signaling in adult neurons during the training period is required for LTM, and (5) 20E induces CREB-mediated transcriptional activation. Together, these results indicate that the steroid molting hormone 20E has a novel, nondevelopmental role in the formation of long-lasting memory in adult insects (Ishimoto, 2009),

The temporal profile of 20E titers during embryonic, larval, and pupal stages is essentially controlled by the genetically determined developmental program. As previously shown, environmental stimuli, such as high temperature and nutritional shortage, induce up-regulation of 20E levels in adult flies. This study has demonstrated that 20E levels are increased in male flies after they are paired with a mated female for 7 h, conditions under which a robust courtship LTM is generated. Ecdysone signaling activated by these environmental stimuli or social interactions may trigger specific molecular and cellular responses in adults, and lead to long-lasting changes in physiology and behavior (Ishimoto, 2009),

In flies, steroid hormone synthesis is known to occur primarily in 2 organs, the larval prothoracic gland and the adult female ovary . Ecdysteroids are present in adult males as well as females. It remains to be determined where ecdysteroids are produced other than in the female ovary, and how their synthesis is regulated in adults. The last 4 sequential hydroxylations of their synthesis, which convert steroid precursors into 20E, are catalyzed by 4 cytochrome P450 enzymes encoded by phantom, disembodied, shadow, and shade, known collectively as the Halloween genes. The temporal changes in ecdysteroid levels during development are mainly attributed to transcriptional regulation of these genes. To understand the regulatory mechanisms for production of ecdysteroids in adult flies, it is important to examine where these enzymes are expressed, and how their expression and activity are regulated. Recent studies show that feeding the dopamine precursor L-DOPA to young Drosophila virilis females increases the dopamine (DA) content in the body, and subsequently results in a substantial increase in 20E levels. Given that dopamine has been implicated in negatively reinforced memory, it is possible that this neurotransmitter acts as a mediator between environmental stimuli and an elevation of 20E level (Ishimoto, 2009),

Using a temperature-sensitive EcR allele and an RNAi that targets EcR, it was shown that courtship LTM is impaired by conditional suppression of EcR function during the training period. Also, LTM was restored in the EcR temperature-sensitive mutants as long as they were maintained at the permissive temperature during the training period. These experiments demonstrate that ecdysone signaling through nuclear EcRs has an important role in the physiological processes that are necessary for the formation of LTM. How does ecdysone contribute to the formation of LTM? One possibility is that fully functional ecdysone signaling is required for effective sensory processing, and that the adverse effect of a 50% reduction in EcR expression on the learning process is due to severe sensory dysfunction. However, this possibility is not likely, because the courtship behavior of male flies with reduced EcR function was fond to be qualitatively and quantitatively comparable with that of control males. Also, EcR/+ males exhibited a short-lasting courtship memory after 1-h training, which suggests that their sensory acuity and ability to acquire courtship memory are rather normal. Thus, it is proposed that ecdysone signaling operates in the CNS, and contributes to consolidation of the memories into a long-lasting form. The MB is considered to be the center of olfactory memory. The EcR RNAi experiments suggest that the MB is one of the brain structures required for the influence of ecdysone on the formation of courtship LTM. Also, the study using the CRE-luc reporter indicates that CREB, a key regulator of long-lasting modifications of the nervous system, is involved in ecdysone-dependent LTM formation (Ishimoto, 2009),

Given that genetically programmed ecdysone signaling is known to control neuronal remodeling during development, it is interesting to speculate that certain experiences may recapture the ecdysone-mediated developmental processes in the adult brain and lead to structural and functional modifications to the nervous system that facilitate the formation of stable, LTM. The ability of ecdysone to remodel the nervous system is known not to be limited to developmental stages. For example, in the adult house cricket (Acheta domesticus) brain, ecdysone has been shown to inhibit proliferation of neuroblasts in the MBs and to trigger their differentiation into interneurons. Although there is no evidence of continued neurogenesis in the adult Drosophila brain, it is possible that ecdysone signaling induces significant changes in properties of existing neurons, resulting in structural and functional remodeling of neuronal circuits. A recent study has shown that the canonical ecdysteroid transcriptional cascade in the MB neurons of the adult worker honey bee (Apis mellifera) is initiated in response to activated ecdysone signaling, further suggesting the involvement of ecdysteroids in remodeling the adult nervous system (Ishimoto, 2009),

These findings in Drosophila indicate that regulation of memory by environmentally induced steroids could be ancient in origin, and widespread in species that have an ability to learn and remember. Thus, the molecular components and signaling pathways responsible for steroid-mediated memory regulation are likely to be shared, at least in part, by evolutionarily diverse animal species. This study has focused on the role of EcRs, nuclear hormone receptors that function through transcriptional regulation of their target genes, in the formation of LTM. Recently, a novel Drosophila G protein-coupled receptor (DopEcR) was found to be activated by ecdysteroids. Thus, it is also interesting to examine the possible involvement of rapid, nongenomic actions of ecdysone in regulation of memory. Considering the relatively simple nervous system of flies, the extensive knowledge of the genetics of this organism, and the highly developed experimental tools available for its study, Drosophila should be an ideal model system to elucidate the molecular, cellular, and neural-circuit bases of memory regulation by steroid hormones (Ishimoto, 2009),

The steroid molting hormone Ecdysone regulates sleep in adult Drosophila melanogaster

Ecdysone is the major steroid hormone in insects and plays essential roles in coordinating developmental transitions such as larval molting and metamorphosis through its active metabolite 20-hydroxyecdysone (20E). Although ecdysone is present throughout life in both males and females, its functions in adult physiology remain largely unknown. This study demonstrates that ecdysone-mediated signaling in the adult is intimately involved in transitions between the physiological states of sleep and wakefulness. First, administering 20E to adult Drosophila promotes sleep in a dose-dependent manner, and it does so primarily by altering the length of sleep and wake bouts without affecting waking activity. Second, mutants for ecdysone synthesis displayed the 'short-sleep phenotype,' and this was alleviated by administering 20E at the adult stage. Third, mutants for nuclear ecdysone receptors showed reduced sleep, and conditional overexpression of wild-type ecdysone receptors in the adult mushroom bodies resulted in an isoform-specific increase in sleep. Finally, endogenous ecdysone levels increase after sleep deprivation, and mutants defective for ecdysone signaling display little sleep rebound, suggesting that ecdysone is involved in homeostatic sleep regulation. In light of the recent finding that lethargus--a period at larval-stage transitions in the nematode worm C. elegans--is a sleep-like state, the results suggest that sleep is functionally and mechanistically linked to a genetically programmed, quiescent behavioral state during development (Ishimoto, 2010).

This study demonstrates that ecdysone is intimately involved in the regulation of Drosophila sleep and that ecdysone has a sleep-promoting effect. These conclusions are based on the sleep analysis using the Drosophila Activity Monitoring (DAM) system. A recent study pointed out that sleep, particularly daytime sleep, could be erroneously defined by the DAM system due to its inability to detect brief movements of flies. To evaluate the sleep phenotype in EcRA483T/EcRNP5219 and Dominant temperature sensitive 3 (DTS-3)/+ flies (DTS-3 displays dominant lethality during development and lowers Ecdysone levels) independently of the DAM system, their movements from ZT4 to ZT8 were directly observed using a video-recording system and sleep parameters were calculated using a Drosophila sleep analysis software, pySolo. The video-based analysis demonstrated that sleep is indeed reduced in EcRA483T/EcRNP5219 and DTS-3/+ flies during the observation period, confirming the original conclusions drawn from the DAM-based sleep analysis (Ishimoto, 2010).

Previous reports have shown that ecdysone signaling at the adult stage plays a role in the regulation of oogenesis, stress responses, life span, and formation of long-term memory. It appears that ecdysone signaling is activated in adults when they are in stressful environments, possibly as a means of urgently managing unfavorable internal conditions caused by these environments. In that sense, ecdysone or 20E might have a function as a stress hormone in adult flies. EcR/+ and DTS-3/+ flies, in which ecdysone signaling is less active, exhibit an increase in life span relative to their wild-type counterparts, suggesting that frequent or chronic activation of ecdysone signaling is detrimental in adults because it alters metabolic states and leads to increases in the generation of harmful by-products. One of the proposed functions for sleep is to remove undesirable by-products that accumulate during the waking state. Interestingly, 20E levels in wild-type flies tend to increase during daytime, possibly corresponding to the generation of harmful by-products during the major waking period. Flies with suboptimal ecdysone signaling sleep less and fail to exhibit adequate sleep rebound following sleep deprivation. These flies may not accumulate detrimental materials to the same extent as their wild-type counterparts, reducing their sleep need (Ishimoto, 2010).

The fact that EcRA483T/EcRNP5219 flies show severe defects in sleep indicates that EcR-mediated gene transcription is important for sleep regulation. However, the results suggest that EcR-independent pathways also play a role in the ecdysone-mediated sleep–wake regulatory processes. Specifically, wake-bout duration is likely controlled by such EcR-independent pathways, in light of the following observations. Wake-bout duration during the day is drastically increased in DTS-3/+ mutants and is considerably decreased in 20E-treated wild-type flies; thus ecdysone signaling has significant effects on wake-bout duration. However, EcRA483T/EcRNP5219 females, in which EcR-mediated ecdysone signaling is severely impaired, display normal wake-bout duration. Moreover, administering 20E to EcRA483T/EcRNP5219 females leads to a significant reduction in wake-bout duration (daytime 34.0% and nighttime 30.8%) as is seen in the 20E-treated control flies. Together, these results suggest that EcR-dependent ecdysone signaling pathways are rather dispensable for the regulation of wake-bout duration. Unlike the EcR-dependent transcriptional cascades, which have been well characterized, little is known about the nature of EcR-independent, 'nongenomic' ecdysone pathways. One potentially important component of the latter pathways is DopEcR, a novel G protein-coupled receptor with structural similarity to vertebrate β-adrenergic-like receptor (Srivastava, 2005). In vitro experiments have demonstrated that the activity of DopEcR can be modulated by both dopamine and ecdysteroids and that DopEcR has effects on multiple intracellular signaling cascades. Future functional studies of DopEcR are expected to provide insights into possible functions of EcR-independent ecdysone signaling in the regulation of sleep and wakefulness (Ishimoto, 2010).

An intriguing hypothesis for the function of sleep is that it contributes to the modulation of synapses in the brain and thus to neural plasticity. Ecdysone has an intrinsic ability to modulate the structure and function of the nervous system during both development and adulthood. It has been shown in Drosophila that ecdysone controls neuronal remodeling during formation of the adult nervous system and that it does so through signaling pathways involving EcRs and TGF-β. Ecdysone also plays a role in remodeling of the adult brain in the house cricket (Acheta domesticus), in this case by inhibiting neuroblast proliferation in the MBs and triggering their differentiation into interneurons. In honeybees (Apis mellifera L.), ecdysone exposure activates an EcR-mediated transcriptional cascade in the adult MB neurons, suggesting that ecdysone is important for reorganization of the adult brain. Moreover, it has recently been shown that long-term courtship memory in Drosophila, which is likely associated with the stable modification of synaptic function and/or structure in the adult brain, is dependent on EcR-mediated ecdysone signaling. These findings are consistent with the possibility that ecdysone is involved in sleep-associated changes to structure and function in the adult brain. A recent study reported that several synaptic marker proteins in the Drosophila brain show widespread alterations in their expression levels as a function of sleep-wake cycles. The data suggest that the endocrine system, in particular ecdysone signaling, contributes to such global changes in the adult nervous system and thus plays important roles in the regulation of the brain states during sleep and wakefulness (Ishimoto, 2010).

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation

Steroid hormones are required in Drosophila for progression of oogenesis during late stages of egg maturation. This study shows that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay switching on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed (König, 2011).

This study shows that in Drosophila ecdysone signalling regulates differentiation of a GSC daughter and modulates ovarian stem cell niche size. The delay in GSC progeny differentiation correlates with reduced expression levels of TGF-β pathway components. Based on expression patterns it appears that germarial somatic cells, niche and ECs are the critical sites of ecdysteroid action and a co-activator of ecdysone receptor, Taiman is the spatially restricted regulator of ecdysone signalling in soma. During adulthood the ecdysone pathway has a specific role in EC differentiation and soma-germline cell contact establishment. In addition, during development the ecdysone signalling pathway has a role in somatic niche formation (König, 2011).

Ecdysteroids in general control major developmental transformations such as metamorphosis and morphogenesis in Drosophila. Different tissues and even different cell types within the same tissue respond to this broad signalling in a specific fashion and in a timely manner. In the developing Drosophila ovary steroid hormone receptors are expressed in a well-timed mode, high levels coinciding with proliferative and immature stages and low levels preceding reduced DNA replication and differentiation. Mutations in ecdysone pathway components affect ovarian morphogenesis, including heterochronic delay or acceleration in the onset of terminal filament differentiation. During the niche establishment the levels of both ecdysone receptors, EcR and USP are greatly downregulated in anterior somatic cells that will contribute to the niche per se. This study shows that perturbation of ecdysone signalling in pre-adult ovarian soma leads to the formation of enlarged niches. The specific response to systemic hormonal signalling in niche precursors is achieved by a specific function of the ecdysone receptor co-activator Taiman. When timely regulation of ecdysone signalling does not occur, more cells are recruited to become niche cells resulting in enlarged niches that are capable to host more stem cells. These data first show that ecdysone steroid hormonal signalling regulates the formation of the adult stem cell niche and suggest that a developmental tuning of ecdysone signalling controls the number of anterior somatic cells that will differentiate into cap cells (König, 2011).

It is logical that stem cell division and germline differentiation are regulated by some systemic signalling depending on the general state of the organism, which depends on age, nutrition, environmental conditions and so on. Hormones are great candidates for this type of regulation as they act in a paracrine fashion and their levels are changing in response to ever-changing external and internal conditions. Steroid binding to nuclear receptors in vertebrates triggers a conformational switch accompanied by increased histone acetylation that permits transcriptional co-activators binding and the transcription initiation complex assembly. In Drosophila, the trithorax-related protein, a histone H3 methyltransferase that like Taiman belongs to the p160 class of co-activators, and an ISWI-containing ATP-dependent chromatin remodelling complex (NURF), that regulates transcription by catalysing nucleosome sliding, both bind EcR in an ecdysone-dependent manner, showing that chromatin modifications can mediate response to this general signalling. Transcriptional regulation has a key role in GSC maintenance and differentiation, for example, the TGF-β ligand Dpp secreted by niche cells induces phosphorylation of the transcription factor Mad in GSCs that in turn suppresses transcription of the differentiation factor Bam. In addition, it has been shown recently that in Drosophila adult GSC ecdysone modulates the strength of TGF-β signalling through a functional interaction with the chromatin remodelling factors ISWI and Nurf301, a subunit of the ISWI-containing NURF chromatin remodelling complex (Ables, 2010). Therefore, it is plausible that ecdysone regulates Mad expression cell autonomously via chromatin modifications. Since pMad directly suppresses a differentiation factor Bam, it is expected that Bam would be expressed in pMad-negative cells. Interestingly, the findings show that ecdysone deficit decreases amounts of phosphorylated Mad in GSCs and also cell non-autonomously suppresses Bam in SSCs. As SSCs that express neither pMad nor Bam are accumulated when the ecdysone pathway is perturbed it suggests that there should be an alternative mechanism of Bam regulation. Even though eventually this still can be done on the level of chromatin modification, the data suggest that the origin of this soma-generated signal may be associated with cell adhesion protein levels. Further understanding of the nature of this signalling is of a great interest (König, 2011).

The progression of oogenesis within the germarium requires cooperation between two stem cell types, germline and somatic (escort) stem cells. In Drosophila, reciprocal signals between germline and escort (in female) or somatic cyst (in male) cells can inhibit reversion to the stem cell state and restrict germ cell proliferation and cyst growth. Therefore, the non-autonomous ecdysone effect can be explained by the necessity of two stem cell types that share the same niche (GSC and ESC) to coordinate their division and progeny differentiation. This coordination is most likely achieved via adhesive cues, as disruption of ecdysone signalling affects turnover of adhesion complexes and cytoskeletal proteins in somatic ECs: mutant cells exhibited abnormal accumulation of DE-Cadherin, β-catenin/Armadillo and Adducin (König, 2011).

Cell adhesion has a crucial role in Drosophila stem cells; GSCs are recruited to and maintained in their niches via cell adhesion. Two major components of this adhesion process, DE-Cadherin and Armadillo/β-catenin, accumulate at high levels in the junctions between GSCs and niche cells, while in the developing cystoblasts and escort cells levels of these proteins are strongly reduced. Levels of DE-Cadherin in GSCs are regulated by various signals, for example, nutrition activation of insulin signalling or chemokine activation of STAT, and this study shows that in ESCs it is regulated by steroid hormone signalling. Possibly, these two stem cell types respond to different signals but then differentiation of their progeny is synchronised via cell contacts. While hormones, growth factors and cytokines certainly manage stem cell maintenance and differentiation, the evidence also reveals that the responses to hormonal stimuli are strongly modified by adhesive cues (König, 2011).

Specificity to endocrine signalling can be achieved via availability of co-factors in the targeted tissue. Tai is a spatially restricted co-factor that cooperates with the EcR/USP nuclear receptor complex to define appropriate responses to globally available hormonal signals. Tai-positive regulation of ecdysone signalling can be alleviated by Abrupt via direct binding of these two proteins that prevents Tai association with EcR/USP (Jang, 2009). Abrupt has been shown to be downregulated by JAK/STAT signalling (Jang, 2009). Interestingly, JAK/STAT signalling also has a critical role in ovarian niche function and controls the morphology and proliferation of ESCs as well as GSCs. JAK/STAT signalling may interact with ecdysone pathway components in ECs to further modulate cell type-specific responses to global endocrine signalling. A combination of regulated by different signalling pathway factors that are also spatially and timely restricted builds a network that ensures the specificity of systemic signalling (König, 2011).

Knowledge of how steroids regulate stem cells and their niche has a great potential for stem cell and regenerative medicine. The current findings open the way for a detailed analysis of a role for steroid hormones in niche development and regulation of germline differentiation via adjacent soma (König, 2011).

Steroid signaling within Drosophila ovarian epithelial cells sex-specifically modulates early germ cell development and meiotic entry

Drosophila adult females but not males contain high levels of the steroid hormone ecdysone, however, the roles played by steroid signaling during Drosophila gametogenesis remain poorly understood. Drosophila germ cells in both sexes initially follow a similar pathway. After germline stem cells are established, their daughters form interconnected cysts surrounded by somatic escort (female) or cyst (male) cells and enter meiosis. Subsequently, female cysts acquire a new covering of somatic cells to form follicles. Knocking down expression of the heterodimeric ecdysteroid receptor (EcR/Usp) or the E75 early response gene in escort cells disrupts 16-cell cyst production, meiotic entry and follicle formation. Escort cells lose their squamous morphology and unsheath germ cells. By contrast, disrupting ecdysone signaling in males does not perturb cyst development or ensheathment. Thus, sex-specific steroid signaling is essential for female germ cell development at the time male and female pathways diverge. These results suggest that steroid signaling plays an important sex-specific role in early germ cell development in Drosophila, a strategy that may be conserved in mammals (Morris, 2012).

These studies show that ecdysone signaling promotes multiple, fundamental steps of early oogenesis. Steroid signaling maintains the structure of the GSC niche and allows somatic niche cells to support a normal rather than a reduced number of GSCs. Subsequently, this pathway promotes 16-cell cyst production, meiotic entry and follicle formation. In contrast, male germ cell development lacks a steroid signaling requirement. Despite the fact that male somatic cyst cells interact with developing male germ cells in a very similar manner as in the ovary, and that male cysts form and enter meiosis like their female counterparts, disrupting steroid production or steroid pathway genes for eight days in these cells caused no detectable effect (Morris, 2012).

Ecdysone signaling was previously reported to be essential for initiating cystoblast development and for cell adhesivity. Germaria from flies in which signaling was reduced using similar methods to those applied in this study accumulated excess single-spectrosome-containing germ cells (cystoblasts). In contrast, no extra cystoblasts were seen unless knock down flies were followed beyond 8 days. The appearance of extra cystoblasts after prolonged gene knock down correlated with extensive alterations in the normal structure of the GSC niche and anterior germarium. The blockade in cystoblast specification/differentiation is therefore likely to be secondary to changes in somatic support cell shape and function, which are required to limit the range of the BMP signals repressing germ cell differentiation. Consequently, it is believed that ecdysone signaling directly affects the processes described here, but is only secondarily involved in cystoblast differentiation (Morris, 2012).

The formation of 16-cell cysts and entry into meiosis are closely linked. Shortly after completing synchronous mitoses that generate a new 16-cell cyst, all the germ cells enter the first meiosis-specific process, pre-meiotic S phase. The strong reduction in meiotic, 16-cell cyst formation that was observed when ecdysone signaling is reduced, suggests that hormones control meiotic entry during Drosophila oogenesis. Meiosis in many lower organisms is induced by nutrient limitation and modulated by nutrient-sensitive pathways Ecdysone signals may help determine when cysts have been starved sufficiently to enter meiosis, much as they assess nutrient sufficiency at other decision points (Morris, 2012).

If steroid signaling in the ovarian soma acts to mediate the extraordinary metabolic demands of female gamete production, then the absence of a male requirement is not surprising. The metabolic demands of egg production are immense, unlike those of sperm production. Thus, decisions affecting oocyte progression may have evolved to employ conserved mechanisms also used during life stage transitions such as dauer formation in C. elegans or the larval/pupal transition. This fundamental difference between male and female gametogenesis may apply to a wide range of organisms and might explain why sex-specific steroid signaling is a common aspect of gametogenesis (Morris, 2012).

Steroid hormone signaling plays a major role in mammalian sex determination and gametogenesis. Transcriptional changes controlled by the Y chromosome-linked SRY gene and hormonal differences dependent on the Sf1 nuclear receptor begin to orchestrate divergent germ cell developmental fates in the bipotential mouse gonad. At this stage, germ cells in both the both male and female gonad are engaged in cyst formation. In females, cysts are completed and enter meiosis while in the testis cyst formation and gamete development arrests. Whether estrogen mediates cyst completion and meiotic entry in female mice in a manner similar to the role of ecdysone in Drosophila remains an interesting question. Squamous, pre-granulosa cells surround mouse germline cysts at the time of follicle formation, and treatment of pregnant animals with estrogen or progesterone enhances the production of multi-oocyte follicles. This raises the possibility that steroid signaling also plays a conserved role during mammalian follicle formation (Morris, 2012)

Ecdysone receptor (EcR) suppresses lipid accumulation in the Drosophila fat body via transcription control

Lipid metabolism drastically changes in response to the environmental factors in metazoans. Lipid is accumulated at the food rich condition, while mobilized in adipocyte tissue in starvation. Such lipid mobilization is also evident during the pupation of the insects. Pupation is induced by metamorphosis hormone, ecdysone via ecdysone receptor (EcR) with lipid mobilization, however, the molecular link of the EcR-mediated signal to the lipid mobilization remains elusive. To address this issue, EcR was genetically knocked-down selectively in 3rd instar larva fat body of Drosophila, corresponding to the adipocyte tissues in mammalians, that contains adipocyte-like cells. In this mutant, lipid accumulation was increased in the fat body. Lipid accumulation was also increased when knocked-down of taiman, which served as the EcR co-activator. Two lipid metabolism regulatory factor, E75B and adipose (adp) as well as cell growth factor, dMyc, were found as EcR target genes in the adipocyte-like cells, and consistently knock-down of these EcR target genes brought phenotypes in lipid accumulation supporting EcR function. These findings suggest that EcR-mediated ecdysone signal is significant in lipid metabolism in insects (Kamoshida, 2012).

The transcription factor Grainy head and the steroid hormone ecdysone cooperate during differentiation of the skin of Drosophila melanogaster

The arthropod epidermis is an epithelium that deposits the apical cuticle, which is a stratified extracellular matrix (ECM) protecting the animal against pathogens, preventing dehydration and also serving as an exoskeleton. Differentiation of the cuticle conceivably implies coordinated production, secretion and localization of its components. The underlying molecular mechanisms are poorly explored. This work shows that the transcription factor Grainy head and the steroid hormone ecdysone drive the production of two partially overlapping sets of cuticle factors. Nevertheless, Grainy head is needed to modulate the expression of ecdysone signalling factors; the significance of this cross-talk is yet unclear. In addition, it was found that ecdysone signalling negatively regulates its own impact. In conclusion, thes findings suggest that at least two independently triggered pathways have evolved in parallel to cooperatively ensure the stereotypic implementation of the cuticle. As Grainy head is also essential for epithelial differentiation in vertebrates, it is speculated that Grainy head acts to decode the ancient skin program common to all animals. Full differentiation of the skin necessitates a second, complementing taxon-specific program that requires its own decoder, which is represented by ecdysone in arthropods, whereas the vertebrate specific one remains to be identified (Gangishetti, 2012).

Ecdysone, which peaks at mid-embryogenesis, acts through the nuclear receptor EcR that in turn activates the expression of several other transcription factors including βFtzF1 during late embryogenesis. Upon interference with EcR function specifically, the transcript levels of both retroactive (rtv) and, unexpectedly, knickkopf (knk), which is insensitive to Shade (Shd) function, dropped. This finding suggests that EcR may have ecdysone-independent functions during cuticle differentiation. Up-regulation of shd transcription in stage 17 embryos with hampered EcR activity suggests that EcR normally suppresses shd expression. To determine whether cuticle differentiation may involve βFtzF1 activity, the phenotype was studied of larvae with impaired βFtzF1 function. Mutations in βftzf1 had a mild effect on cuticle differentiation that resembles those provoked by mutations in rtv, serp and verm. Consistently, the expression of those genes depending on ecdysone, ie rtv, pot, serp and verm, was also sensitive to βFtzF1 activity, whereas expression of knk was unaffected by mutations in βftzf1. As the expression of doublesex cognate 73 (dsc73), which depends on Shd, is independent of βFtzF1, it is concluded that the ecdysone pathway runs only partially through βFtzF1 to regulate cuticle differentiation. Possibly, for full cuticle differentiation the ecdysone pathway has to be slowed down at the end of embryogenesis, a mechanism that is triggered by ecdysone itself as, like EcR, βFtzF1 represses shd transcription. Efficient transcriptional response to ecdysone signalling is also influenced by Grh, which contributes to full ftzf1 expression. Hence, regarding its impact on ecdysone signalling, Grh is a factor with antipodal functions: on the one hand it impedes the synthesis of ecdysone by repressing the expression of shd, and on the other, as an antagonist of Shd, it supports the expression of βftzf1, thereby enforcing the ecdysone response. Thus, taken together, Grh intervenes with the ecdysone pathway by modulating the balance of Shd and βFtzF1 activity. Why is the expression of βFtzF1 targets not suppressed in grh mutant stage 17 embryos? Again, Grh may control timed progress of ecdysone signalling including βFtzF1 function without influencing the amplitude of target expression. Alternatively, redundant function of ecdysone mediators such as E75 and E74 may take over the role of βFtzF1 for a robust ecdysone response in grh mutant animals (Gangishetti, 2012).

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. This syudy shows that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. In a simpler model system, this study used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. In response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells (Regan, 2013).

Using an in vivo genetic approach to block EcR signaling specifically in hemocytes, this study has shown that ecdysone directly regulates their cell shape. Moreover, the data indicates that ecdysone regulates the onset of hemocyte motility and dispersal at metamorphosis, reflecting its function in border cell motility during oogenesis. Microarray data reveal that EcR up-regulates the expression of several genes functioning in cell motility or cell shape regulation, which could account for these phenotypes. Arguably, migration of hemocytes between tissues is required for clearing dying larval tissues during the pupal period. Hemocytes expressing the EcRDN construct do not engulf dead cells, which is potentially a consequence of impaired phagocytosis, motility, or a combination of both, although it is not possible to distinguish between these possibilities. Ecdysone has previously been shown to induce the expression in the hemocyte-derived mbn2 cell line of croquemort (crq), a gene encoding a receptor for apoptotic cells in the embryo. crq was identified in the microarray analysis as showing EcR-dependent up-regulation at metamorphosis, and this was confirmed by qPCR, where crq expression is almost completely suppressed in EcRDN-expressing pupal hemocytes. The impaired expression of crq in EcRDN hemocytes likely contributes to their deficiency in apoptotic cell phagocytosis. Functionally, the regulation of hemocytes by ecdysone, which is the coordinator of larval tissue apoptosis, may be a smart way for the fly to synchronize its macrophage scavenging activity with the moment it is most needed, at metamorphosis. Surprisingly, no gross developmental consequences were observed of the loss of this function, whereby HmlΔGal4>EcRB1DN individuals completed metamorphosis without delay. This is in agreement with studies showing that under sterile conditions, pupae lacking hemocytes altogether progress normally through metamorphosis. It suggests that dead cells might be engulfed by other, non-professional phagocytes (e.g. neighbor cells as reported for tumorigenesis), cleared up by other unidentified means, or simply tolerated, in the absence of functional hemocytes (Regan, 2013).

Furthermore, it was show that the activation of hemocyte motility at metamorphosis also correlates with a change in their response to induced epithelial damage. While in the larva hemocytes are passively recruited to wounds from circulation, this study demonstrates that in the pupa they actively migrate to damaged tissues. Induction of epithelial wounds at different times APF demonstrated that active wound responsiveness is progressively acquired at metamorphosis. In agreement with previous ex vivo analysis, the current data highlights an intriguing plasticity of hemocytes to adapt their migratory activity and their response to wounds throughout development: chemotaxis in embryos and pupae versus passive circulation and ‘capture’ to wounds in larvae. This correlates with the observation that, although the heart is beating in a 20 h APF-old pupa, hemocytes are not propelled in the hemolymph by the heartbeat, but maintain a slow, steady, active migration on tissues (Regan, 2013).

Most importantly, this study provides the first in vivo evidence of hormonal regulation of the Drosophila cellular response to bacterial challenge. With both ex vivo and in vivo data, this study has demonstrated an important role for EcR in the up-regulation of hemocyte phagocytic activity at metamorphosis. How does ecdysone signaling regulate phagocytosis? Previous studies in hemocyte-derived cell lines have shown that ecdysone treatment increases the transcription of some immune-related genes encoding AMPs and immune receptors such as Crq. Using a tissue-specific, whole genome transcriptomic approach, this study demonstrates that many genes are regulated by ecdysone signaling in hemocytes at metamorphosis. This analysis reveals the molecular regulation behind the observed phenotypes and allows for the identification of candidate effector genes. For example, 35 genes up-regulated by EcR at metamorphosis have been previously attributed a function in phagocytosis. These genes encode proteins involved in different steps of the phagocytosis process, such as recognition (e.g. the receptors PGRP-LC, croquemort, and Nimrod family members, Dscam and scab), or cytoskeletal rearrangements required for the engulfment step (e.g., RhoGAP71E, Rac2, Arpc5 and SCAR). Interestingly, PGRP-LC (FC 1.8 by microarray, 3.9 by qPCR) was recently shown to be induced in ecdysone-treated S2 cells. It appears that ecdysone can regulate the phagocytosis process at different levels, which may be necessary to co-ordinate the ability of hemocytes to recognize and engulf their target. Moreover, genes regulated by ecdysone signaling can be implicated in more than one process, for example phagocytosis and AMP expression (e.g. PGRP-LC), or phagocytosis and cell migration (e.g. SCAR); this may contribute to synchronisation of different hemocyte immune functions (Regan, 2013).

The functional relevance of increased cellular immune activity at metamorphosis is an intriguing question. Recent studies of the contribution of cellular immunity to Drosophila defenses have revealed that flies in which hemocytes are genetically ablated present a high lethality at metamorphosis. This is likely the result of opportunistic bacterial infections, as feeding antibiotics was sufficient to restore wild-type viability. No such lethality was observed under normal conditions when expressing EcRDN in hemocytes; Phagoless lethality in absence of infection is also lower than that previously described . This suggests that the fly strains and fly food used in this study do not harbor the same bacterial types as those used in previous studies, leading to distinct opportunistic infection scenarios. Nevertheless, these data indicate a significant lethality of HmlΔ>EcRDN pupae not only after septic injury with E. faecalis or E. carotovora, but also after oral infection at larval stages with E. carotovora, a bacterium that is not usually lethal in wild-type individuals. This lethality is quite dramatic considering only hemocytes express the transgene, and is similar to the lethality in hemocyte-ablated individuals . It indicates that ecdysone regulation is essential for hemocyte immune functions and survival after infection (Regan, 2013).

Metamorphosis may represent a stage of predisposition to opportunistic oral infection, as the larval midgut is replaced by the adult intestinal epithelium. It is speculated that histolysis of the gut could release bacteria from the lumen into the body cavity; active hemocytes may be required to limit the spreading of bacteria from temporary weak points in the epithelium. HmlΔ>EcRDN prepupae induce a normal intestinal and systemic humoral immune response after being orally infected at larval stage. In the case of both septic injury and oral infection, it is therefore likely that the main cause of decreased survival in HmlΔ>EcRDN pupae is their striking hemocyte phagocytosis phenotype, possibly in combination with lack of motility, inability to chemotax to damaged tissue or other potential uncharacterized hemocyte defects (Regan, 2013).

The synchronization of multiple processes is a fundamental requirement for successful development, and likely to rely on hormonal signaling. Altogether, the current data reveal the importance of steroid hormone signaling in the synchronization of development and immunity in Drosophila, by ecdysone-dependent activation of hemocytes at pupariation. it has been have recently shown that ecdysone signaling affects the humoral response through regulation of PGRP-LC expression. Interestingly, an impact of this regulation was obsered on the ability of adult flies to survive infection, indicating that ecdysone regulation of immunity extends beyond metamorphosis. In humans, hormonal activation of macrophages underpins various cancer pathologies and is therefore highly relevant in clinical terms. It is also generally accepted that steroid hormones impact immunity in mammals. For example, glucocorticoids are commonly used in pharmacology for their anti-inflammatory properties. However, their regulation of the immune response is complex, as they can also enhance the immune response. More generally, steroid hormones' specific action on monocytes is still not very well documented, mainly due to the complexity of mammalian systems and experimental limitations. Elucidating mechanisms for steroid hormone regulation of cellular immunity will be essential for a full understanding of sex differences in immunity and inflammation (Regan, 2013).


Ecdysone receptor: Biological Overview | Evolutionary homologs | Regulation | Targets of Activity | Protein interactions | Developmental Biology | References

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