Ecdysone receptor

Targets of Activity (part 2/2)

Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila

The ecdysone response hierarchy mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates (Buszczak, 1999).

In order to investigate the role of ecdysone-responsive gene expression in the ovary, expression of three classical early ecdysone-responsive genes, E75, E74 and BR-C were examined. In situ hybridization revealed that the E75 and E74 genes are transcribed in remarkably similar patterns during oogenesis. Both E75 and E74 transcripts are first detected in region 2b of the germarium. Expression decreases during stages 2-4 and low levels of E75 and E74 mRNA are again detected in stage 5-7 egg chambers. Transcription of E75 and E74 appears to be upregulated during stage 8 in both the germline and soma. This expression continues to increase until stage 10B when transcription of both genes peaks in the follicle cells and the nurse cells. Immunofluorescent staining reveals the presence of BR-C protein in the follicle cell nuclei beginning between stages 5 and 6 of oogenesis. In most of the egg chambers examined, BR-C appears to be completely absent from the germline. However, in rare cases, low levels of expression could be detected in the nurse cell nuclei. These observations are consistent with a recent report that describes follicle cell expression of BR-C mRNA (Buszczak, 1999).

The expression of E75, E74 and BR-C in egg chambers suggests that these genes are co-regulated by a common signal. If these early response genes are being regulated by ecdysone, one would expect a dependence on the ecdysone receptor. To determine whether the ecdysone receptor is present in the ovary, egg chambers from Canton-S females were stained using anti-EcR antibodies. Antibody staining reveals that germline and somatic cells express EcR protein in their nuclei. This expression is first detected in the germarium, appears to be slightly upregulated during stage 4 and persists until the late stages of oogenesis. Additionally, border cells strongly express EcR during their migration through the nurse cell cluster. Uso has also been detected in all cells within the ovary. Thus, both components of the functional ecdysone receptor are present in the germline and soma during all stages of oogenesis (Buszczak, 1999).

To test the dependence of early response gene expression on ecdysone, a study was made of the effects in ovaries of the ecdysoneless¹ (l(3)ecd¹) mutation, in which low levels of ecdysone are generated. Females homozygous for the temperature-sensitive mutation l(3)ecd¹ lose the ability to lay eggs after just 2 days at the restrictive temperature and to have 13% of the wild-type ovarian ecdysone titer when shifted to the restrictive temperature for 4 days. E75 transcript levels were compared in wild-type and l(3)ecd¹ females shifted to the restrictive temperature for different lengths of time. Using in situ hybridization, no difference in E75 mRNA levels could be detected between ovaries taken from wild-type and l(3)ecd¹ females maintained at 25°C. However, there is a reproducible reduction of E75 mRNA in l(3)ecd¹ ovaries relative to wild-type controls shifted to the restrictive temperature for 2 days. An RNAse protection assay was used to quantitate the difference in E75 transcription in l(3)ecd¹ and wild-type ovaries. This analysis reveals that l(3)ecd¹ ovaries contain approximately half the E75 mRNA of wild-type ovaries when subjected to restrictive conditions. BR-C expression in wild-type and l(3)ecd¹ ovaries was also assayed. Immunofluorescent staining showed that BR-C protein levels appear to be reduced in l(3)ecd¹ ovaries relative to wild-type controls. A reduction of BR-C expression in ovaries from mutants shifted to 29°C was also detected on Western blots (Buszczak, 1999).

The presence of EcR protein and the presence of USP in ovarian cells of all stages suggests that these cells are competent to respond to ecdysone. A test was performed to see whether an increase in the ecdysone titer could induce E75 expression in the ovary. Ovaries were cultured in the presence or absence of 20-hydroxyecdysone. In situ hybridization shows that E75 transcription increases in early egg chambers in response to ecdysone and that the increase of expression occurs in both the follicle cells and germline. An RNAse protection assay was used to quantitate the induction of E75 transcription by exogenous ecdysone. This analysis has demonstrated that increasing amounts of ecdysone in the culture media leads to increased expression of E75 (Buszczak, 1999).

During stage 10, the follicle cell expression of E75 becomes enriched in the dorsal anterior cells. This suggested that inputs in addition to ecdysone are needed to refine E75 expression. Previous work has shown that follicle cell polarity is established during mid- to late-oogenesis and depends on the interaction between Gurken and the Drosophila homolog of the mammalian EGF receptor (Egfr). To determine whether E75 expression is under control of the dorsoventral signaling pathway, ovarian E75 mRNA distribution was examined in dorsalized and ventralized mutant backgrounds. In fs(1)K10 mutants, mislocalization of Grk protein results in activation of Egfr in all anterior follicle cells surrounding the oocyte. In fs(1)K10 mutant egg chambers, E75 expression expands to a ring of anterior follicle cells surrounding the oocyte. Mutations in Egfr prevent signal transduction by the receptor and lead to the ventralization of the eggshell and embryo. In situ analysis indicates that stage 10 follicle cells overlying the oocyte in Egfr mutants no longer express E75. However, E75 expression in the nurse cells is unaffected. These experiments show that the Egfr signaling pathway regulates E75 expression in the dorsal follicle cells but not in the germline (Buszczak, 1999).

To investigate the functional role of E75 in the germline, germline clones of a strong E75 allele were generated using the FLP/FRT system of mitotic recombination and the ovoD1 dominant female sterile transgene. While heterozygotes carrying a control chromosome over ovoD1 laid eggs following clone induction, females carrying the E75 e213 mutation in trans to the ovo D1 chromosome did not lay eggs after clone induction. Upon dissection, E75 e213 germline clones appear to arrest and degenerate at stages 8-9. Some yolk can be seen in the oocyte but the follicle cells rarely complete their migration to the oocyte. While the germline in these clonal egg chambers degenerates, the follicle cell layer remains remarkably intact. Oocytes in E75 e213 germline clones are small and sometimes displaced to one side of the posterior end of the egg chamber. The frequency of the observed phenotypes strongly suggests that E75 function is required in the germline to progress beyond early vitellogenesis. If E75 expression depends upon ecdysone response, similar phenotypes should result from ecdysone receptor mutations. To test whether EcR is required for the completion of oogenesis, germline clones of the EcR null mutation, EcR M554fs, were analyzed. A small number of females laid eggs 2-4 days after eclosion. Although few in number (2- 3/female/day), these eggs appear to be normal in shape and size. However, egg laying ceases after 4-5 days. Upon dissection, ovaries from several females (15/202) appear to have clonal egg chambers many of which are arrested at stage 6 or 7. These egg chambers are easily distinguished from non-clonal ovo D1 egg chambers by the lack of EcR antibody staining in nurse cell nuclei. Therefore, it appears as though EcR is required for egg chamber progression beyond mid-oogenesis (Buszczak, 1999).

In order to address more directly the timing and autonomy of steroid hormone signaling in the Drosophila ovary, the expression pattern and germline requirements for the recently described dare gene were analyzed (Freeman, 1999). dare encodes the Drosophila homolog of adrenodoxin reductase, an enzyme required for the synthesis of steroid hormones in vertebrates. Biochemical studies from insects, and genetic analysis of the dare locus in Drosophila, strongly suggest that insect steroid hormone production is also critically dependent upon adrenodoxin reductase, and null alleles of dare appear to effectively block the synthesis of ecdysteroids (Freeman, 1999). In situ hybridization reveals that low levels of Dare mRNA can be detected in the germline beginning at stage 6 of oogenesis. Expression of dare remains relatively low until stage 10 when egg chambers exhibit a dramatic increase of dare transcription. To further assess the requirements for dare during oogenesis, germline clones using the dare 34 allele (Freeman, 1999) were induced. While all of the control females laid eggs, very few females with presumptive dare 34 clones lay eggs, and these females lose the ability to lay eggs after 1-2 days. These findings indicate that dare has a critical germline function, and are consistent with the possibility that dare clones arrest at a point similar to that seen for other components of the ecdysone response pathway. Interestingly, significant dare expression is not seen in the germarium; while lower levels of dare could potentially be sufficient to allow some ecdysone synthesis, this raises the possibility that signals other than ecdysone could contribute to germarial expression of early response genes (Buszczak, 1999).

In other insects, including the mosquito Aedes aegypti, the transition from the previtellogenic to the vitellogenic state is governed by ecdysone-regulated hierarchies. Aedes egg chambers develop synchronously and remain arrested in a previtellogenic state until the female takes a blood meal. This triggers production of the ecdysiotropic neuoropeptide (EDNH) that stimulates ovarian synthesis of ecdysone. The resulting increase in the ecdysone titer leads to the expression of E75 and controls induction and progression of vitellogenesis and further egg development (Pierceall, 1999). In a similar fashion, ecdysone could be regulating egg chamber progression past stage 8 in Drosophila. Thus, the stage-specific expression of ecdysone response hierarchies and their control over egg chamber development may represent an evolutionarily conserved mechanism for coordinating the developmental processes that occur during insect oogenesis. While the synchronous development of a cohort of eggs under endocrine control, as seen in the mosquito, would not be unexpected, the asynchronous progression of Drosophila oogenesis under similar hormonal control raises an interesting and important question. How can a presumed endocrine factor, such as ecdysone, regulate the sequential, asynchronous induction of these genetic regulatory programs during Drosophila oogenesis? Stage specificity of ecdysone-responsive gene expression could, in principle, be controlled either at the level of competence to respond to ecdysone or at the level of the production or availability of the hormone itself. The results presented here suggest the latter possibility. While analysis of EcR germline clones indicates that the receptor is required during mid-oogenesis, the ecdysone receptor is present throughout oogenesis. The expression of the ecdysone response genes BR-C, E74 and E75, in contrast, is stage-specific, and varies in conjunction with experimental manipulation of hormone titer. These findings suggest that individual egg chambers are exposed to different amounts of hormone as they progress through oogenesis (Buszczak, 1999).

Microarray analysis of Drosophila development during metamorphosis

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA⁺ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

Relatively little is known about basic metabolic processes during metamorphosis. Most work has focused on the alcohol dehydrogenase gene, which is known to be repressed by ecdysone, but a few studies have included the ecdysone-inducible glucose dehydrogenase gene and the ecdysone-repressible urate oxidase locus. All three of these genes behave as expected. Nine genes encoding enzymes in the glycolytic pathway are present on the array and are down-regulated during the late larval ecdysone pulse. Also, there are reduced expression levels of genes encoding enzymatic constituents of the citric acid cycle, oxidative phosphorylation, amino acid metabolism, fatty acid oxidation and synthesis, glycogen synthesis and breakdown, and the pentose phosphate pathway. Thus, some tissues must be responding to the initiation of metamorphosis by tempering their metabolic activity. This reduction may represent an early response in certain tissues that are destined to undergo programmed cell death, or it may reflect a global response to the transition from an active larva to a sessile prepupa (White, 1999).

Gene expression changes during metamorphosis also foreshadow both larval muscle breakdown and adult myogenesis. At approximately 2 hours APF, the anterior larval musculature begins to break down. This breakdown lasts until approximately 6 hours APF. Genes encoding both structural and regulatory components of muscle formation are down-regulated as early as 4 hours BPF (see Muscle-specific genes regulated during metamorphosis). In addition to the repression of genes encoding components of thin and thick filaments, genes encoding other muscle-specific molecules are also repressed, including factors that compose the mesh in which these filaments lie and regulatory factors involved in the specification of muscle tissue. The mRNAs of all these repressed genes decrease substantially many hours before histolysis of the anterior larval muscles and therefore predict the occurrence of this morphological event well before it begins. Twenty-four hours APF, adult myogenesis is well underway. The genes DMef-2, bagpipe, and tinman are all up-regulated at 12 hours APF from the baseline at PF, coincident with the prepupal pulse of ecdysone. It is suggested that induction of these regulatory factors initiates the development of the adult musculature, which will establish itself several hours later (White, 1999).

In contrast to the histolyzing larval muscles, the CNS undergoes dramatic differentiation and restructuring during early metamorphosis. The majority of the CNS is composed of adult-specific neurons that reorganize at this time by extending processes and establishing new connections. Several genes known to be involved in neuronal-specific processes are differentially regulated during the late larval ecdysone pulse (see Developmental control genes induced during metamorphosis.) For example, the Drosophila neurotactin and plexin A genes are induced. These genes are involved in axonal pathfinding and in establishing synaptic connections. The neurotactin (nrt) gene product is involved in growth cone guidance and is localized to the cell surface at points of interneuronal cell contact in the presumptive imaginal neurons within the larval CNS. Nerve cord condensation does not occur normally in the late third instar CNS of nrt mutant animals. In prepupae, nrt is expressed in a tissue- and cell type-specific manner: it is restricted to a small set of ocellar pioneer neurons in the brain, photoreceptors of the eye, and some sensory neurons in the developing wing. It is suggested that nrt, like the control genes induced from >18 hours BPF to PF, is regulated by the late larval ecdysone pulse. The plexin A gene belongs to a family of genes that encode Ca²⁺-dependent homophilic cell adhesion molecules first identified in the vertebrate CNS and PNS. Drosophila Plexin A also acts as a receptor for class I semaphorins, and both loss of function and overexpression experiments demonstrate that Plexin A is involved in axon guidance and repulsion of adjacent neurons (defasciculation). Many neurons defasciculate in response to ecdysone during nervous system remodeling, and it is suggested that an increase in plexin A expression may be partly responsible for this response. Several more differentially expressed neuronal-specific molecules are shown at The Drosophila Microarray Project. These genes provide several new candidates for factors that are involved in the neuronal outgrowth and morphological remodeling responses to ecdysone (White, 1999).

Larval-specific tissues such as the aforementioned larval muscles, the midgut, and the salivary glands undergo programmed cell death during metamorphosis. Genes involved in programmed cell death were identified in these experiments. The apoptosis-activating reaper gene has previously been shown to be ecdysone-inducible, and this is reflected in the data. Expression of the Drosophila caspase-1 gene is also observed during the prepupal ecdysone pulse but not during the late larval pulse. This gene is also an activator of apoptosis, and mutants display melanotic tumors and larval lethality. Induction of a cell death inhibitor gene, thread (also known as Diap1), is observed during the late larval pulse but not the prepupal phase. The DIAP1 protein includes inhibitor-of-apoptosis (IAP) domains and has been identified as a factor that can block reaper activity. Because different tissues begin apoptosis at different stages of development, changes in the expression of inhibitors and activators of apoptosis are expected to be tissue-specific. For example, the expression profiles observed for the caspase-1 activator and the Diap1 inhibitor are those expected in tissues such as the larval salivary glands. Tissue-specific information on the induction of these genes will be important to an understanding of the coordination of apoptosis during metamorphosis (White, 1999).

The expression levels of genes involved in cellular differentiation also dynamically change during metamorphosis. The gene headcase is expressed in all proliferating imaginal cell lineages. This gene is induced during the prepupal ecdysone pulse but does not substantially change expression levels during the late larval ecdysone pulse. Imaginal tissues in headcase null mutants appear normal in size and shape but fail to differentiate normally. These mutants are invariably pupal lethal and show pleiotropic effects in adult tissues. The predominant headcase loss of function phenotype is defective head development. Mutants can display deletion of the head capsule, leaving only a protruding proboscis. Another gene expressed in this manner with a known role in ecdysone-mediated differentiation of imaginal discs is IMP-L2, an essential secreted immunoglobulin family member implicated in neural and ectodermal development in Drosophila. These data demonstrate that factors required for cellular differentiation during metamorphosis are present in the data set. There are 29 other EST sequences encoding novel genes that display a greater than threefold induction from PF to 12 hours APF but do not display a threefold or greater change in expression level during the late larval ecdysone pulse. Perhaps some of these genes, such as headcase and IMP-L2, are involved in differentiation of adult-specific tissues (White, 1999).

Other genes known to be involved in cellular differentiation exhibit changes in level of expression during metamorphosis. For example, corkscrew is induced during the late larval ecdysone pulse. This gene encodes a protein tyrosine phosphatase that is involved in receptor tyrosine kinase signaling during photoreceptor differentiation. shortsighted encodes a bZIP transcription factor homologous to a mouse TGF-beta-responsive gene and acts in the decapentaplegic pathway. This gene is induced during the late larval ecdysone pulse and then further induced during the prepupal pulse. tolkin encodes a TGF-beta homolog and is induced during the late larval ecdysone pulse but not during the prepupal pulse. tolkin is expressed in imaginal discs during metamorphosis and causes pupal lethality when mutated. These results establish potential connections between known signal transduction pathways and ecdysone-initiated metamorphosis (White, 1999).

Description of wild-type development is a first step in understanding metamorphosis from a global perspective. However, it is of interest to understand the composition of the genetic hierarchy that leads to metamorphosis. To test whether new targets of transcription factors could be identified in the ecdysone genetic hierarchy, the ecdysone-induced nuclear receptor DHR3 was prematurely expressed at >18 hours BPF. DHR3 is responsible for the coordination of part of the transcriptional program controlling metamorphosis and can act as either a repressor or an activator of transcription, depending on the target gene. DHR3 can induce betaFTZ-F1, a nuclear receptor that is active during midprepupal development and is responsible for the difference in the genetic response to ecdysone between the late larval and prepupal ecdysone pulses. betaFTZ-F1 induction is confirmed by the microarray results. Several other genes are induced by DHR3 when it is expressed at >18 hours BPF. One of these is represented by a novel EST (LD24139) that is induced from 3 to 9 hours APF during wild-type development. ESTs representing 12 other DHR3-induced genes that have less than threefold induction at 3 to 9 hours APF are listed at DHR3-regulated genes. Some of these additional genes may not normally be DHR3 targets or may be induced by DHR3 at other stages during development (White, 1999).

DHR3 has been shown to inhibit the induction of ecdysone-inducible genes, and with E75B it can act as a repressor of the betaFTZ-F1 gene. DHR3 is expressed before the ecdysone-inducible genes are up-regulated but is still capable of repressing genes. Four out of seven such genes belong to the cytochrome P450 (CYP) class of genes. Three of these CYP genes are normally repressed during the late larval pulse, and this repression begins before DHR3 induction occurs (approximately 4 hours BPF). Thus, DHR3 cannot be solely responsible for their repression, although it may contribute to it. One function of cytochrome P450 molecules is hydroxylation of steroids; the depletion of transcripts of the CYP genes may provide a mechanism by which production of the biologically active form of ecdysone (20-hydroxyecdysone) is stifled at PF. Regulation of these CYP genes within the ecdysone hierarchy further suggests that they may have a role in controlling the ecdysone genetic cascade (White, 1999).

Taken together, these results demonstrate the utility of DNA microarrays in determining the genetic foundations of metamorphosis. The identities of the differentially expressed genes discovered in this study suggest several points of coordination between the ecdysone-regulated pathways that control the temporal aspects of metamorphosis and the developmental pathways that control the specification and differentiation of particular cell types and tissues. Despite the experimental restrictions imposed by the use of whole animals, changes have been observed in the abundance of transcripts that correlate with the late larval or prepupal ecdysone pulses (or both) for genes whose activities have not been known to be influenced by this hormone. Further studies are now needed to delineate the relationship between the ecdysone-regulated genetic hierarchies and the functions of both the known and the novel genes that are differentially expressed during metamorphosis. For example, a next step would be to distinguish genes that are directly regulated by ecdysone from those that are secondary targets of ecdysone-regulated factors (White, 1999).

Data produced on a genomic scale can be used to similarly assist in deciphering the complex genetic networks that control other stages of Drosophila development. Great strides have of course been made in defining these networks by use of mutations and expression constructs. The resulting stick diagrams describing these networks must, however, be incomplete, in part because inactivation of the majority of genes does not result in obvious mutant phenotypes. Genomic approaches have the potential to expand these stick diagrams to include all functional genes. Integrating and visualizing data derived from genomic studies present a substantial challenge. Nonetheless, combining the powerful molecular and genetic approaches that Drosophila offers with genomic information will inevitably produce a reasonably complete picture of gene regulation and its implications for metazoan development (White, 1999).

Tissue-specific gene expression and Ecdysone-regulated genomic networks in Drosophila

During insect metamorphosis, each tissue displays a unique physiological and morphological response to the steroid hormone 20-hydroxyecdysone (ecdysone). Gene expression was assayed in five tissues during metamorphosis onset. Larval-specific tissues display major changes in genome-wide expression profiles, whereas tissues that survive into adulthood display few changes. In one larval tissue, the salivary gland, a computational approach was used to identify a regulatory motif and a cognate transcription factor involved in regulating a set of coexpressed genes. During the metamorphosis of another tissue, the midgut, genes encoding factors from the hedgehog, Notch, EGF, dpp, and wingless pathways are activated by the ecdysone regulatory network. Mutation of the ecdysone receptor abolishes their induction. Cell cycle genes are also activated during the initiation of midgut metamorphosis, and they are also dependent on ecdysone signaling. These results establish multiple new connections between the ecdysone regulatory network and other well-studied regulatory networks (Li, 2003).

Developmental patterns of gene expression were studied from five different tissues and organs: central nervous system (CNS), wing imaginal disc (WD), larval epidermis and attached connective tissue (ED), midgut (MG), and salivary gland (SG), during late larval and early prepupal development when ecdysone triggers metamorphosis. At these stages of development, the five tissues display very different morphological and physiological responses to ecdysone. The wing imaginal disc responds to the hormone by initiating evagination, or unfolding, as it changes from a compact epithelial bilayer to an extended appendage. The salivary glands secrete glue proteins that are used to immobilize the puparium during metamorphosis. The cuticle attached to the larval epidermis undergoes a process of hardening and tanning to form the pupal case. The central nervous system (CNS) displays little morphological change during the late third instar ecdysone pulse, but the animal displays changes in behavior and in neurosecretory status. The two major types of cells in the larval midgut, larval epidermal cells and adult epidermal progenitor cells (midgut imaginal islands), respond in opposite ways to ecdysone. The larval epidermal cells initiate the process of programmed cell death, while the imaginal cells proliferate and form the adult midgut (Li, 2003).

One tissue, the midgut, was selected to assay during its complete metamorphosis, which occurs from 18 hr before puparium formation (BPF) to 12 hr APF. During this 30 hr period, eleven time points were examined as the larval midgut is destroyed and replaced with the adult midgut. The two major cell types present in this organ are distinguishable by size. The larval epithelial cells are large, with decondensed polyploid nuclei, and undergo programmed cell death in response to ecdysone. Embedded among the larval cells are small diploid imaginal midgut cells, which proliferate in response to the hormone to form the adult epithelial cells. Additionally, the midgut contains relatively small numbers of muscle, tracheal, and endocrine cells (Li, 2003).

In total, transcripts from a surprisingly large fraction of the genome, >30%, changed significantly during the metamorphosis of the midgut (18 hr BPF to 12 hr APF). Broad classes of temporally separable gene expression patterns are evident. These classes include sets of transcripts that rapidly decrease coincident with onset of programmed cell death in the larval cells, sets that are induced during early or late metamorphosis, and sets of transcripts expressed at highest levels during the middle period of the time course when the larval cells are in the final stages of cell death and the adult cells are rearranging to form new tissue (Li, 2003).

Within these broad classes, specific sets of genes that have related functions and show parallel expression were identified, indicating that they make up gene batteries. Six such examples, included coregulated transcripts that encode proteins found in specific macromolecular complexes, biochemical pathways, organellar functions, and structural components of the cells that compose this tissue. Transcripts encoding proteasome components increase during the ecdysone pulse that triggers the onset of cell death in larval cells. Transcripts encoding glycolytic enzymes rapidly decrease during the initiation of metamorphosis, but gradually resume expression as the imaginal cells proliferate. Vacuolar ATPases shows a pattern similar to the glycolytic enzymes, whereas tubulin- and actin-encoding transcripts peak during the intense period of imaginal cell proliferation and migration as the adult midgut is formed. Transcripts encoding structural components of the peritrophic membrane of the mature larval gut gradually decrease during its replacement with adult tissue (Li, 2003).

The expression patterns were examined of regulatory genes known to be involved in the ecdysone transcriptional hierarchy predicted to control the gene batteries that were identified. Also examined was the expression of genes with known roles in programmed cell death or cell cycle control. The expression of known ecdysone-responsive regulatory genes was consistent with previous observations in midgut. Although the larval midgut is composed of cell types that undergo divergent responses to ecdysone -- apoptosis and cell proliferation -- it was nonetheless possible to detect significant changes in transcript levels from genes encoding proteins involved in both processes. The apoptosis activator gene ark was expressed at 4 hr BPF. E93 and reaper, which encode proteins that serve as critical control points in the commitment to programmed cell death, were expressed at PF, as was the initiator caspase dronc. These midgut expression profiles were compared to those reported for salivary glands at and after 10 hr APF, when a prepupal pulse of ecdysone triggers apoptosis in that tissue; almost the entire genetic cascade was found to be similarly activated in salivary glands and midgut albeit at two distinct periods of development. However, one notable difference was observed at the top level of the cascade. In the salivary gland, E93 is activated by βFTZ-F1, whereas in the midgut the βFTZ-F1 gene is not induced until 6-8 hr after E93 is induced. The regulation of E93 therefore does not depend on βFTZ-F1 in the midgut, but must rely on another as yet unidentified factor(s). During midgut metamorphosis, developmental modulation of transcript levels were also observed for genes encoding DNA polymerases, cyclins, CDCs, and other cell cycle regulators, as well as genes encoding DNA repair proteins such as Hus1, Rad23, and PCNA/Mus209 (Li, 2003).

Which of the genes that are differentially expressed at the onset of midgut metamorphosis require ecdysone signaling? Ecdysone-dependent transcriptional activity was removed using mutant Ecdysone Receptor (EcR) alleles, rescuing null EcR mutants to the third larval instar by using a heat shock-inducible EcR transgene. Gene expression was examined in mutant midguts that were isolated from mutant animals arrested at the end of the third larval stage (stage 2a mutants). 376 (76%) of the 495 genes that are significantly induced during the onset of midgut metamorphosis (18 hr BPF to 2 hr APF) required EcR function, whereas 296 (64%) of 460 transcripts that decline significantly in level during this time period require ecdysone signaling through EcR. Thus, a very large proportion of the genes that are developmentally regulated at the initiation of metamorphosis in this organ are under the control of the transcription factors that mediate the ecdysone signal. However, it does not appear that EcR function is a general requirement for transcription, because a significant fraction of differentially expressed genes are unaffected in EcR mutant tissue (Li, 2003).

Of the several different classes of genes expressed during midgut metamorphosis, the regulation of all genes in the proteasome, tubulin/actin, and lysozyme clusters requires EcR to exhibit their normal changes in developmental expression. However, many genes in the v-ATPase cluster and nearly half the genes in the peritrophin cluster did not require EcR. The downregulation of hexokinase A, 6-phosphofructokinase, and pyruvate kinase genes in the glycolysis pathway were affected in the EcR mutants, while many others in this pathway were not. Hexokinase A, 6-phosphofructokinase, and pyruvate kinase are rate-controlling enzymes in the glycolytic pathway, indicating that their ecdysone dependence is functionally significant. The expression of the numerous known ecdysone receptor target genes such as E75, E74, broad, E23, and DHR3 required EcR as expected. The induction dynamics for the E74 and DHR3 transcription factor genes was as expected, as was their dependence on EcR. In contrast to E74 and DHR3, DHR78 has previously been described to reside upstream of EcR at the top of the ecdysone regulatory hierarchy -- the expression of EcR is dependent on the wild-type function of DHR78. However, DHR78 can also be induced by ecdysone in organ culture. The results demonstrate that DHR78 wild-type induction is indeed dependent on EcR function. Taken together, these data indicate a positive feedback loop between EcR and DHR78 during the onset of metamorphosis in the midgut (Li, 2003).

Genes encoding factors involved in cell cycle and growth control, and in DNA repair, are also under the control of EcR. In spite of the role of ecdysone in stimulating cell proliferation during metamorphosis, no cell cycle genes have previously been linked to the ecdysone regulatory hierarchy. The induction of the cell cycle regulatory genes CyclinB, cdc2, and CyclinD were all observed to be dependent on EcR function. The rapid induction of cdc2 during the late third instar ecdysone pulse is similar to that observed for direct targets of EcR. The CyclinD gene is also induced at this time, but its maximal induction occurs several hours after that observed for cdc2. Cyclin D promotes cellular growth, whereas Cyclin B/Cdc2 controls G2/M transitions in proliferative cells. The dependence of these three genes on EcR function indicates that ecdysone may control cell proliferation, at least in part, through their regulation. Coordinate with the induction of CyclinB, cdc2, and CyclinD, the induction was observed of DNA polymerase-delta and DNA repair genes such as Rad23, and PCNA/mus209. The induction of these DNA repair and synthesis genes is also EcR dependent. The expression changes of these genes may be the result of the direct action of EcR, or due to the action of factors directly controlled by the ecdysone receptor complex. It is unlikely that the increase in expression of these genes is simply due to increased numbers of proliferative cells because the total number of divisions between 18 hr BPF and PF are few, and not all cell cycle or DNA repair genes showed an increase in expression at the initiation of metamorphosis. For example, the level of CyclinJ, which is known to be required during early embryonic division cycles, is actually reduced in expression from 18 hr BPF to PF. When the expression of cell death genes was examined in EcR mutant tissue, E93 induction was observed as well as induction of the Ark caspase activator and the dronc caspase gene required wild-type function of EcR (Li, 2003).

Factors in several well-studied signaling pathways are induced during midgut metamorphosis. These include Wnt (dishevelled, armadillo, and zeste white 3), TGFβ/BMP (sara, daughters against dpp, and glass bottom boat), EGFR (torpedo/egfr, rhomboid/veinlet, vein, and Keren/spitz2), and Notch pathway genes (delta, kuzbanian, suppressor of hairless, E(spl)malpha, and E(spl)mβ). All of these pathways are used during embryonic midgut development, and these data indicate they are reused during midgut metamorphosis. Genes in the Hedgehog signaling pathway (hedgehog, smoothened, and cubitus interruptus) changed significantly as well (Li, 2003).

To determine whether any of the genes in these pathways are expressed as a consequence of ecdysone signaling, the EcR mutant expression data was examined for those genes that were induced during the late third instar ecdysone pulse. The induction of zeste white-3/shaggy, keren/spitz2, kuzbanian, and hedgehog are all dependent on the presence of functional EcR. The induction dynamics of the EGFR ligand gene keren/spitz2, the Notch proteolytic activation factor gene kuzbanian, and the shaggy/zeste white-3 kinase gene are similar to genes that are known direct targets of ecdysone signaling. The induction of hedgehog follows a secondary response pattern, as do genes from the E(spl) complex that are induced in response to Notch activation, although these induction kinetics are also consistent with these genes being partially activated directly by the ecdysone receptor and partially with other factors (i.e., they may be 'early-late' genes). These data show that the regulatory network controlled by ecdysone in midguts includes the activation of known components of the Wnt, EGFR, Hedgehog, and Notch pathways. Notably, ligand production for the EGF, Hedgehog, TGFβ/BMP, and Notch pathways is under control of ecdysone. The specific roles that each of these pathways plays during metamorphosis are currently unknown. These results nonetheless indicate new connections between ecdysone signaling and the activity of several other signaling pathways during the metamorphosis of this organ, either through direct targeting of the ecdysone receptor or through the actions of downstream factors (Li, 2003).

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. This study reveals a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicates that Ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies show that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo (Rusten, 2004).

The fat body is a primary nutrient-responsive tissue that emulates the functions of the liver and adipose tissue of vertebrates. Fat body cells undergo programmed autophagy during the last larval stage (L3) preceding pupariation. Findings in Mamestra brassicae have demonstrated that autophagy can be induced by ecdysone. In Drosophila, autophagy is developmentally upregulated from mid-L3 stage in fat body cells. Although previously the levels of ecdysone during the L3 stage of development in Drosophila have been detected only at low levels at the wandering-L3 stage and increase markedly before puparium formation in most studies, a small hormonal peak before the initiation of wandering has been reported. Expression of dominant-negative Ecdysone receptor from the mid-L3 stage under Lsp-Gal4 control results in a dramatic reduction of autophagy. The autophagic area is severely reduced, and fewer acidic structures are observed. Since complete inhibiton of autophagy was not observed, the timing of Lsp-Gal4 expression was closely followed and it was found that expression initiated reporter gene expression 20-30 min after programmed autophagy had been started. Most likely, this latency of Lsp-Gal4 expression accounts for the incomplete penetrance of the phenotypes. In fact, driving expression of the dominant-negative EcR using a constitutive fat body Gal4 driver (cg-Gal4), or placing a temperature-sensitive mutant of ecdysoneless (ecd¹) to restrictive temperature at the start of the L3 stage, led to a complete inhibition of autophagy. This suggests that ecdysone has a regulatory role on programmed autophagy already at the early L3 stage and that the ecdysone titer at this stage of development is at the threshold of detection. The issue of ecdysone levels during larval development has recently been revisited: radioimmunoassay measurements detected low but continuously increasing levels of ecdysone during mid-L3 stage. Taken together, these results suggest that programmed autophagy is due to this low but rising level of ecdysone during the L3 stage of development (Rusten, 2004).

A simple explanation of ecdysone-induced programmed autophagy could be the initiation of wandering and therefore starvation-induced autophagy. This is not likely, however, for the following reasons. Developmental autophagy is initiated in fat bodies of late feeding animals at least 6 hr before the animals stop feeding and leave the food, and 12 hr before ingested food starts to disappear from the anterior part of the midgut. In addition, the autophagic response in fat body cells is uncoupled from the change in feeding behavior since expression of a dominant-negative ecdysone receptor cell autonomously inhibited programmed autophagy in late wandering L3 animals that ceased feeding 8 hr earlier (Rusten, 2004).

PI3K signaling is able to regulate autophagy and is unlikely to be a part of the amino acid sensing mechanism during an acute starvation response; amino acids and insulin have been shown to control autophagic proteolysis through different signaling pathways in rat hepatocytes. This is supported by the fact that the presence of the PIP₃ binding probe, GFP-PH, at the cell membrane is not affected by amino acid deprivation in the fat body. The loss of PI3K signaling was only observed after 24 hr of starvation, long after the acute starvation response of autophagy. This concurs with observations in cultured mammalian cells in which insulin signaling and PI3K activity do not respond to variations in nutrient levels (Rusten, 2004).

What is then the physiological significance of PI3K signaling regulating autophagy? PI3K signaling was modulated in the fat body to see if it could influence the autophagic response to ecdysone. Elevation of PI3K signaling during the period of programmed autophagy prevented the biogenesis of autolysosomes. This epistatic regulation of PI3K signaling over ecdysone-induced programmed autophagy suggests that PI3K signaling is a part of the same pathway or a dominant repressor. A strong reduction and ultimately loss of PI3K signaling was observed in the fat body during the induction of programmed autophagy, suggesting that ecdysone downregulates PI3K signaling. In addition, a reduction in PI3K signaling failed to increase the autophagic activity during programmed autophagy, in line with the idea that it is in the same pathway and is already inhibited completely. In contrast, reducing Tor signaling in the fat body could further increase autophagic activity during developmental autophagy, suggesting that Tor is not inhibited completely, or not involved in programmed autophagy. Elucidating the role of Tor signaling during this process requires further studies (Rusten, 2004).

Several lines of evidence support a role for PI3K signaling in ecdysone-induced programmed autophagy in the fat body. (1) Inhibition of Ecdysone receptor activity or an increase in PI3K signaling produced very similar phenotypes, indicating that these pathways perform opposite regulatory roles on programmed autophagy. (2) Administration of the ecdysone analog RH5849 to feeding larvae promoted attenuation of PI3K signaling. (3) Clonal inactivation of ecdysone receptor signaling led to a failure of this attenuation. (4) Simultaneous downregulation of PI3K signaling and inhibiton of ecdysone receptor activity restored programmed autophagy to wild-type levels. Thus, ecdysone signaling is both necessary and sufficient for downregulation of the PI3K pathway during programmed autophagy. Taken together, these results suggest a model in which ecdysone receptor signaling has the ability to promote autophagy through the downregulation of PI3K signaling (Rusten, 2004).

A Drosophila insulin-like peptide promotes growth during nonfeeding states

In metazoans, tissue growth relies on the availability of nutrients—stored internally or obtained from the environment—and the resulting activation of insulin/IGF signaling (IIS). In Drosophila, growth is mediated by seven Drosophila insulin-like peptides (Dilps), acting through a canonical IIS pathway. During the larval period, animals feed and Dilps produced by the brain couple nutrient uptake with systemic growth. This study shows that during metamorphosis, when feeding stops, a specific DILP (Dilp6) is produced by the fat body and relays the growth signal. Expression of DILP6 during pupal development is controlled by the steroid hormone ecdysone. Remarkably, DILP6 expression is also induced upon starvation, and both its developmental and environmental expression require the Drosophila FoxO transcription factor. This study reveals a specific class of ILPs induced upon metabolic stress that promotes growth in conditions of nutritional deprivation or following developmentally induced cessation of feeding (Slaidina, 2009).

Growth relies on the ability of cells and organisms to access nutrients. Nutrients can be obtained from diverse sources, such as from the environment through feeding, or from internal stores as with early embryos that develop from large eggs. Accordingly, because alternate sources of nutrients are used during specific periods of development, organisms must be able to adapt their metabolic and growth programs to changes in the developmental or environmental energy context (Slaidina, 2009).

In complex animal species, growth is controlled by intermingled paracrine and endocrine regulatory processes, with organ and tissue growth governed by specific genetic programs that determine the target size and relative proportions of the species. The output of these genetic programs is further modified by environmental cues, including nutrition. Variations in nutritional input can influence growth and metabolism via insulin/IGF signaling (IIS). In particular, when nutrients are abundant, IIS is maximally active and growth is limited solely by the organ-intrinsic program; upon nutrient shortage, in contrast, IIS becomes limiting and restricts the growth and metabolic parameters accordingly (Slaidina, 2009).

In mammals, the IIS system is split into two complementary and interacting subsystems that govern growth, metabolism, reproduction, and longevity. The first of these corresponds to circulating insulin levels, which control carbohydrate and fat metabolism, and the second is the GH/IGF-I axis, which regulates cell and tissue growth. Starvation lowers circulating IGF-I, in part through decreased transcription of the IGF-I gene in the liver; this suggests that one major way in which starvation can affect growth is by reducing levels of circulating growth factors (Slaidina, 2009).

The function of IIS in growth control is remarkably conserved in insects, and in particular in Drosophila, where seven Drosophila insulin-like peptides (DILPs) have been identified. The various DILP genes are expressed in different larval and adult tissues, suggesting that they carry nonredundant functions. In particular, DILP1, -2, -3, and -5 are expressed in specialized neurosecretory cells located in each brain hemisphere, called the insulin-producing cells (IPCs). Genetic ablation of these cells leads to severe larval growth deficits, hypertrehalosemia, and increased lifespan (Slaidina, 2009).

One major role for IIS in insects is to couple growth with the animal's energy status. Indeed, total nutrient deprivation downregulates DILP3 and DILP5 transcription in the IPCs, although DILP2 expression remains unchanged. Recent results indicate that variations in nutritional information are relayed by a nutrient sensor operating in the fat body, a larval organ that shares metabolic functions with the vertebrate white fat and liver. In particular, it has been shown that amino acid restriction triggers fat body-specific inhibition of the TOR complex1 (TORC1), a major cell-based nutrient-sensing pathway. Inhibition of TORC1 in the fat body systemically reduces larval growth in part by blocking Dilp secretion from the brain IPCs. Therefore, in line with the decreased levels of circulating IGF-I in vertebrates, starvation affects Drosophila growth by severely reducing brain-specific DILP function (Slaidina, 2009).

Interestingly, previous work has shown that protein starvation causes the growth arrest of endoreplicative larval tissues (ERTs), while only slowing the growth and proliferation of cells in the larval brain and in imaginal discs. Similarly, generally reduced TOR signaling in the larva, which in many respects mimics the starvation state, strongly inhibits ERT growth, while generally sparing the imaginal tissues (ITs) that form the adult structures. This suggests a protection mechanism whereby, under adverse nutrition conditions, the fat body allows larval resources to be reallocated to high-priority tissues like the imaginal discs. Significantly, such a mechanism would require that some ILPs are produced during starvation and activate IIS in the tissues that continue to grow (Slaidina, 2009).

Feeding arrest is also a programmed event during development. At the end of the larval period, animals undergo a stereotyped behavior called the wandering stage, when they migrate away from the food and prepare for pupariation. This developmentally induced starvation precedes the long pupal feeding arrest. During pupal development, larval tissues undergo intense remodeling. This process involves a major reallocation of resources, as future adult tissues form from ITs in a process that uses either nutrient stores that had accumulated in fat cells during larval life, or energy obtained from the degradation of obsolete larval tissues. Since organisms do not feed during this stage, no global growth or weight gain is observed; nevertheless, because tissue remodeling involves cell growth and proliferation, growth-promoting pathways presumably come into play. The paradox of pursuing a growth program in a nonfeeding organism that is subjected to catabolic regulation could be circumvented by the induction of growth-promoting hormones upon feeding arrest (Slaidina, 2009).

This study presents the characterization of a particular DILP, DILP6, which promotes growth during nonfeeding stages. The DILP6 gene is expressed in fat body cells and is strongly induced during the wandering larval and pupal periods, as well as upon starvation. Reduced DILP6 function results in a growth deficit during pupal development and an increased sensitivity to starvation in young adults. The sudden increase of DILP6 expression at the onset of pupal development requires an endocrine signal that is provided by the steroid hormone ecdysone. In parallel, starvation increases DILP6 expression through dFoxO-mediated feedback regulation of IIS. Therefore, DILP6 constitutes an IGF-like peptide with a specialized role in promoting growth during developmentally or environmentally induced nonfeeding states (Slaidina, 2009).

During the successive stages of development, organisms use alternate sources of nutrients to support tissue growth and morphogenesis. In Drosophila, embryonic tissues develop using maternal stores accumulated in the egg in the form of yolk. Larval development follows, with a major growth program relying on the animals' capacity to obtain nutrients from the environment. Finally, during the pupal stage, animals do not feed, and a large quantity of nutrients stored in fat cells allows pupae to prolong growth and finalize the development of adult structures. On top of these basic developmental strategies, feeding larvae have evolved additional buffering mechanisms to protect growing tissues from sudden variations in environmental energy supplies. Notably, brain ILPs promote larval growth and allow the coupling of growth to nutritional input. Their expression and secretion from brain IPCs decrease upon starvation, and several brain DILPs show only residual expression in the pupa. Therefore, there must be a distinct set of growth inducers that take the lead to activate growth in the pupa and upon nutritional stress. In both of these contexts, a physiological switch takes place that triggers the activation of DILP6, a member of a distinct class of ILPs devoted to growth during nonfeeding periods (Slaidina, 2009).

The DILP1 and DILP3 genes are also expressed during pupal development, suggesting that they may act in concert with DILP6. Individual knockout of either of these two DILP genes produces only marginal growth defects, suggesting that there is a high level of redundancy between them or with DILP6. The observation that DILP1 expression increases two-fold in DILP6 mutant larvae suggests a possible compensatory mechanism that could partially suppress the growth impairment observed in DILP6 mutants. The functional class of ILPs represented by DILP6 may be conserved in other insect species, as an ecdysone-induced, fat-body-specific ILP has recently been described in Bombyx mori (Slaidina, 2009).

The developmental and environmental induction of DILP6 involves overlapping mechanisms. First, in response to nutrient deprivation, the IIS component, dFoxO, provokes a burst of DILP6 transcription, thereby linking DILP6 expression with the nutritional status of the animal. This represents a feedback regulation on IIS, as dFoxO, an inhibitor in the IIS pathway, induces the expression of DILP6, an activator of IIS. Interestingly, expression of DILP3 in the adult was also recently shown to depend on dFoxO function, suggesting that other DILP genes in this subclass are subjected to similar controls (Slaidina, 2009).

DILP6 does not appear to be effective as a paracrine/autocrine factor for fat cells. Indeed, fat cells of starved larvae, which express high levels of DILP6, undergo extensive autophagic transformation, even though autophagy has been shown to be blocked in these cells by IIS activation. In addition, overexpression of DILP6 in the fat body of starved larvae does not prevent autophagy (Slaidina, 2009).

More generally, ERTs present stronger growth inhibition in response to starvation than do ITs. The role of a starvation-specific ILP that is induced upon nutritional stress could be to reroute energy stores toward high-priority organs and tissues, such as those responsible for the formation of the future adult. The specific action of DILP6 on imaginal cells could contribute to this diversified behavior, although this would require that ITs are more receptive to the DILP6 signal than are ERTs, at least upon starvation. Such differences in the response of ERTs and ITs to the DILP signal, combined with the production of specific DILPs upon starvation, could constitute a bona fide mechanism for the specific allocation of spare resources to ITs under nutritional stress. However, the mechanisms for such a biased response need to be elucidated (Slaidina, 2009).

At the end of larval development, animals stop feeding and prepare for pupal development. This study shows that tissue remodeling in the pupa involves IIS-dependent growth, and that DILP6 is specifically expressed and required for growth during this period. The transition from larval to pupal development is controlled by the steroid hormone ecdysone (20E), and it was also shown that 20E is required for proper DILP6 induction at the larval/pupal transition. In view of the absence of obvious EcR/Usp binding sites in the 5' region of the DILP6 gene, as well as a previous demonstration that EcR signaling controls dFoxO nuclear localization, it is hypothesized that dFoxO could mediate the ecdysone-dependent expression of DILP6. However, both genetics and ex vivo experiments on dissected fat bodies indicate that, although dFoxO appears to contribute to the developmental induction of DILP6 at the larval/pupal transition, it is not required for the 20E-induced expression of DILP6. In an accompanying manuscript, Okamoto (2009) reports that 20E-induced expression of DILP6 is not affected by cycloheximide, suggesting that the transcriptional induction of DILP6 by EcR/Usp is direct (Slaidina, 2009).

It has been previously shown that ecdysone has a growth-inhibitory function during larval development. Indeed, increased basal levels of circulating ecdysone in larvae can reduce the growth rate and, conversely, decreased basal ecdysone levels can increase the growth rate. Although the mechanisms underlying this relationship are not yet fully understood, this study has established that the levels of ecdysone produced experimentally in these experiments remain close to basal levels, and are insufficient to modify DILP6 expression. Therefore, while basal levels of ecdysone can inhibit systemic growth through an unknown mechanism, high ecdysone levels at the larval/pupal transition can induce DILP6, and thus systemically activate IIS (Slaidina, 2009).

One puzzling observation reported in this study is that the modification of DILP6 expression in pupae can alter the adult mass as well as the resistance of animals to starvation at eclosion. How can DILP6 overexpression in pupae increase adult mass if the mass of the pupa is fixed at the end of larval life? One possible explanation is that DILP6 participates in a tradeoff between the construction of adult tissues and the maintenance of energy stores in the pupa. Indeed, the levels of both TAG and glycogen stores in the young adult are affected by DILP6 levels in the pupa. In this line, recent reports indicate that, under optimal conditions, not all nutrients are used by the pupa, and part of the energy is conserved to provide sustenance during the early period of adult life that precedes feeding. Some larval fat body cells are still present in early adults, and provide energy until feeding begins. Suppressing the death of these cells increases the energy stores and enhances the resistance of young adults to starvation (Aguila, 2007). DILP6 knockdown in pupae has a similar effect: less energy is used by the pupa to build tissues, meaning that the adult ecloses with a smaller body, but with greater energy stores to help overcome early nutritional stress. DILP6 overexpression has the opposite effect. The current results therefore indicate that DILP6 sets the energy balance in pupae by promoting tissue growth, while sparing an energy pool that can be used by the young adult (Slaidina, 2009).

DILP6 shares some specific features with vertebrate IGF-I that distinguish both of them from insulin. DILP6 peptide sequence does not present obvious cleavage sites for an internal C peptide (Brogiolo, 2001). It is produced in the fat body, a tissue sharing common functions with the vertebrate liver, where IGF-I is mainly produced. DILP6 mutant animals present growth defects without obvious metabolic changes, suggesting that DILP6 might have an exclusive growth function. Finally, the induction of growth factor production under conditions of energy stress is also relevant to cancer biology. Indeed, IGF-I and IGF-II are frequently expressed within neoplastic tissue. It is suspected that they act as autocrine and paracrine growth factors within tumors, allowing tumor cells to evade nutritional shortage and acquire survival properties (Pollak, 2008). The induction of DILP6 under starvation and its preferential targeting to ITs instead of ERTs could represent an interesting parallel to the induction of IGFs in tumor cells, where the selective action of growth factors can promote growth and survival of specific tissues in a nonfavorable environment (Slaidina, 2009).

A fat body-derived IGF-like peptide regulates postfeeding growth in Drosophila

Members of the insulin family of peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. This study sshow that Drosophila insulin-like peptide 6 (DILP6), which is structurally similar to vertebrate insulin-like growth factor (IGF), is predominantly expressed in the fat body, a functional equivalent of the vertebrate liver and adipocytes. This expression occurs during the postfeeding stage under the direct regulation of ecdysteroid. dilp6 mutants show growth defects during the postfeeding stage, that result in reduced adult body size through a decrease in cell number. This phenotype is rescued by fat body-specific expression of dilp6. These data indicate that DILP6 is a functional, as well as a structural, counterpart of vertebrate IGFs. The data provide in vivo evidence for a role of ILPs in determining adult body size through the regulation of postfeeding growth (Okamoto, 2009).

DILP6, one of seven ILPs in Drosophila, is produced primarily in the fat body to regulate postfeeding growth without affecting the timing of metamorphosis. This observation is interesting to consider in light of previous findings that suggest that insulin/IGF signaling (IIS) affects both the timing of metamorphosis and the rate of growth. These results thus clearly demonstrate that different ILPs have distinct temporal roles during development. Similar results are also presented in a second publication (Slaidina, 2009; Okamoto, 2009 and references therein).

Insects utilize larval accumulated nutrients for the development of adult-specific tissues during the postfeeding period. How DILP6 mediates this tissue-specific growth remains unknown, but previous reports indicate interplays between 20E and IIS involved in this process. In the fat body, 20E antagonizes IIS, which probably blocks an autocrine effect of DILP6. In contrast, 20E synergistically enhances IIS in the imaginal disks to promote growth. It is also interesting to note that the downregulation of IIS by 20E in the fat body activates autophagy, which promotes the release of stored nutrients. It is suggested that these tissue-specific effects of 20E on IIS facilitate the directional transfer of nutrients from storage organs (fat body) to developing disks to promote adult-specific tissue growth. DILP6 appears to play a pivotal role in this process, and its loss leads to enhanced excretion of unused materials during wandering and after eclosion (Okamoto, 2009).

The independent role of DILP6 compared to IPC-derived DILPs is reminiscent of the roles of IGFs compared to insulin in mammals. There are three major aspects of their similarities. First, it was shown that dilp6 is predominantly expressed in the fat body, a functional equivalent of the mammalian liver and adipose tissue, and the liver is the principal source of circulating IGFs in mammals. Second, the data revealed that the expression of dilp6 is directly regulated by the steroid hormone, 20E, when growth is independent of extrinsic nutritional input. Although the expression of IGFs can be regulated by nutrition, high concentrations of IGF-I and -II are observed during pubertal and fetal development, respectively, reflecting their importance in these key developmental transitions in mammals. Moreover, igf-I expression in several organs is induced by sex steroids, further supporting the analogy between DILP6 and IGFs. Third, the predicted peptide structure of DILP6 is distinct from other DILPs in that it has a short C peptide, which is more similar to vertebrate IGFs than to insulin. Moreover, the short C peptide is likely to remain in the mature form like IGFs, because of the lack of a cleavage site. Thus, the structural aspect also favors the analogy between DILP6 and IGFs. From all these similarities between DILP6 and IGFs, it is proposed that DILP6 is a functional as well as a structural counterpart of vertebrate IGFs, and therefore DILP6 is defined as a Drosophila IGFLP. It should be noted here that, in parallel with the analogy between DILP6 and IGFs, there are several analogies between IPC-derived DILPs and insulin in terms of the source tissues and the nutritional regulation of the expression and peptide secretion (Okamoto, 2009 and references therein).

Together with a previous characterization of IGFLP in Bombyx, it is highly likely that IGFLP is widely present in divergent insect orders. Surprisingly, however, phylogenetic analysis supports no orthology between BIGFLP and DILP6, suggesting that BIGFLP and DILP6 have evolved independently. It is hypothesized that, in ancestral insect species, there was a single ILP that was expressed both in the brain IPCs and in the fat body. This ancestral ILP was probably under distinct regulatory mechanisms (nutritional and developmental) in these tissues, which facilitated functional diversification of IPC-derived ILPs and fat body-derived ILPs after a gene duplication event(s) that happened independently in each insect order. In the previous study in Bombyx, it was demonstrated that BIGFLP is released as a single-chain polypeptide, despite having two potential cleavage sites within the C domain. This suggests the lack of processing enzymes to generate mature insulin in the fat body, which probably explains why fat body-derived ILPs in different species have attained similar structural features as IGFLPs (shortened C-peptide and/or the loss of cleavage sites) despite their independent lineages. Studies in orthopteran species (which are considered closer to earlier insect species), where there is only one identified ILP the expression of which is differentially regulated in the brain IPCs and in the fat body, support the hypothesis (Okamoto, 2009).

Since most insect genomes contain a single insulin/IGF-like receptor gene, IGFLPs and the other ILPs presumably activate the same receptor, although its binding affinities for different ligands likely vary according to the distinct structural features of the ligands. In contrast, mammalian genomes contain multiple receptors, each of which responds to one primary ligand. Therefore, there also appears to exist a clear difference between mammalian IGFs and insect IGFLPs. Considering the pivotal role of IGFs/IGFLPs during development in both of these animal groups, further investigations of the similarities as well as the differences in these signaling pathways should enrich the understanding of underlying mechanisms that control development throughout the animal kingdom (Okamoto, 2009).

Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function

Steroid hormones and their nuclear receptors drive developmental transitions in diverse organisms, including mammals. This study shows that the Drosophila steroid hormone 20-hydroxyecdysone (20E) and its nuclear receptor directly activate transcription of the evolutionarily conserved let-7-complex (let-7-C) locus, which encodes the co-transcribed microRNAs miR-100, let-7 and miR-125. These small RNAs post-transcriptionally regulate the expression of target genes, and are required for the remodeling of the Drosophila neuromusculature during the larval-to-adult transition. Deletion of three 20E responsive elements located in the let-7-C locus results in reduced levels of let-7-C microRNAs, leading to neuromuscular and behavioral defects in adults. Given the evolutionary conservation of let-7-C microRNA sequences and temporal expression profiles, these findings indicate that steroid hormone-coupled control of let-7-C microRNAs is part of an ancestral pathway controlling the transition from larval-to-reproductive animal forms (Chawla, 2012).

This study presents a series of data indicating that the let-7-C locus is a direct transcriptional target of EcR in vivo. The ~2.5 kb primary let-7-C transcript (pri-let-7-C) is detected during the mid-third larval transition, a developmental stage when pulses of 20E activate the transcription of inducible genes through the EcR/Usp nuclear hormone heterodimer. It was found that pri-let-7-C is rapidly induced in cultured Drosophila cells by 20E, and that the 20E responsiveness of the let-7-C locus requires EcR and is mediated by three 13-nucleotide EcR/Usp-binding sites. The deletion of these three EcREs eliminates let-7-C locus expression in larval and pupal tissues, including salivary glands and imaginal discs. EcRE deletion also causes a delay in let-7-C miRNA expression, as well as a reduction of let-7-C miRNA levels in adult flies, and is associated with known let-7-C mutant phenotypes. Taken together, these data strongly suggests that EcR binds to the endogenous let-7-C locus and activates its transcription in response to 20E, and that this transcriptional regulation is required for let-7-C miRNA function (Chawla, 2012).

This work describes a clear convergence in the molecular mechanisms that control developmental timing in Drosophila and C. elegans. Members of the let-7 and miR-125 families of miRNAs were originally identified in C. elegans as part of a pathway of heterochronic genes that promote stage-specific cell fate decisions. This study has directly linked the fly orthologs of these heterochronic genes to the 20E/EcR pathway, which triggers stage-specific transcriptional cascades that direct major developmental transitions, including molting and metamorphosis. One essential function of the 20E/EcR pathway is to activate the expression of let-7-C miRNAs at the end of larval development to promote the formation of adult morphologies required for adult function (Chawla, 2012).

Previous studies showing the slow onset of let-7 and miR-125 expression in tissue culture cells treated with Ecdysone had suggested that hormonal regulation of let-7-C expression may be indirect and not involve the 20E/EcR pathway. This study resolved this issue, showing that pri-let-7-C is detected within 30 minutes of 20E treatment in an EcR-dependent fashion and thus is a likely direct target of 20E/EcR. The delayed expression of processed miRNAs previously observed might be due to the effects of Ecdysone on factors involved in the processing of pri-let-7-C, a possibility that has not been investigated in this study. An unresolved question, however, is why EcR knockdown initiated approximately 18 hours before puparium formation had no effect, in a previous study, on the onset of let-7 and miR-125 expression. It is now thought that let-7 and miR-125 were processed from pri-let-7-C transcripts that were already present when EcR levels were depleted in that experiment. Experiments presented in this study, including analysis of EcR-deficient cell lines and EcR-dominant negative transgenes, indicate that EcR is required for let-7-C expression in vivo (Chawla, 2012).

Some nuclear hormone receptors both activate and repress the expression of direct targets. The C. elegans DAF-12 hormone receptor, for example, activates the miR-241 promoter in the presence of DA steroid hormone, whereas it represses the miR-241 promoter in the absence of DA. Evidence supporting an analogous repressor function has been reported for EcR. The current results suggest that EcR solely functions to activate let-7-C expression in salivary gland and imaginal discs, as removal of EcREs 1-3 results in complete elimination, rather than derepression, of reporter expression in those tissues. The situation is a little less clear in the late larval CNS, as removal of the EcREs reduces but does not eliminate lacZ expression there. This EcRE-deleted reporter is not precociously expressed, though, suggesting that the EcRE sites are not required for reporter repression. It is therefore suspected that let-7-C is co-activated in the CNS by EcR and at least one additional 20E-dependent factor. Indeed, the Sgs3 and Sgs4 salivary glue genes are controlled by complex ecdysone response units, which contain binding sites for other tissue-specific transcription factors such as the homeotic forkhead gene and potential binding sites for Broad-Complex gene (Chawla, 2012).

A growing number of papers have suggested the functional orthology between the 20E/EcR and DA/Daf-12 pathways, as they play similar roles in regulating developmental timing, physiology, reproductive maturation and longevity in flies and worms, respectively. These pathways not only play analogous functions but also share upstream components: the production of 20E as well as DA involves the TGFβ and IGF pathways. The work presented here suggests that they also share at least one common effector: let-7-C miRNAs. Steroid hormone regulation of let-7-C miRNAs may therefore represent an ancestral pathway that plays widespread roles both developmentally and post-developmentally (Chawla, 2012).

Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin

Steroid hormones act as important developmental switches, and their nuclear receptors regulate many genes. However, few hormone-dependent enhancers have been characterized, and important aspects of their sequence architecture, cell-type-specific activating and repressing functions, or the regulatory roles of their chromatin structure have remained unclear. This study used STARR-seq (Self-Transcribing Active Regulatory Region sequencing), a recently developed enhancer-screening assay, and ecdysone signaling in two different Drosophila cell types to derive genome-wide hormone-dependent enhancer-activity maps. The STARR-seq vector couples the candidates' activities to their sequences in cis, such that active enhancers transcribe themselves and are present among cellular RNAs. This setup allows the assessment of candidates independent of whether they are associated with endogenous enhancer-derived transcripts (eRNAs) and irrespective of their locations at or near promoters or transcription start sites (TSSs) or in transcribed regions (e.g., introns or exons), enabling genome-wide enhancer screens, Enhancer activation was shown to depend on cis-regulatory motif combinations that differ between cell types, and cell-type-specific ecdysone targeting can be predicted. Activated enhancers are often not accessible prior to induction. Enhancer repression following hormone treatment seems independent of receptor motifs and receptor binding to the enhancer, as was shown using ChIP-seq, but appears to rely on motifs for other factors, including Eip74. This strategy is applicable to study signal-dependent enhancers for different pathways and across organisms (Shlyueva, 2014).

In this study a quantitative genome-wide map was obtained of hormone-dependent enhancer activity using STARR-seq, a direct activity-based method for enhancer identification. The availability of hundreds of hormone-activated enhancers allowed the systematic dissection of their sequence features, revealing characteristic motif signatures that are predictive within strict cross-validation settings (i.e., when the sequences used for training and testing are strictly separated). These successful predictions mean that the motif signatures are shared across different enhancers and sufficiently general to predict previously unseen sequences not used for training (Shlyueva, 2014).

Interestingly, ecdysone-induced enhancers do not only contain the EcR motif but are also strongly enriched in motifs of putative partner TFs, which differ between cell types and are required for enhancer function. The insufficiency of the EcR to activate transcription and the strict dependence on additional cell-type-specific factors is an important prerequisite to achieve cell-type-specific transcriptional responses via combinatorial regulation. It has been observed for individual transcriptional enhancers that depend on different signaling pathways (Shlyueva, 2014).

Such strictly combinatorial function has been termed 'activator insufficiency' and proposes that ligand-activated TFs function combinatorially with tissue and cell-specific TFs that act as competence determinants and/or coactivators. In this study, the combination of STARR-seq and computational sequence analyses allowed identification of the motif combinations required for ecdysone-activated enhancer function in two different cell types without prior knowledge regarding the hormone receptor and/or putative partners (Shlyueva, 2014).

Repression of enhancer activity after ecdysone treatment appears to be independent of EcR motifs and receptor binding but seems to involve Eip74 motifs. Interestingly, Eip74 had previously been proposed to repress a subset of secondary ecdysone targets, because late puffs in salivary glands appeared larger in Eip74 mutant flies. Because the Eip74 motif mutant enhancer is also less strongly active in the absence of ecdysone, this could mean that Eip74 competes with an activator or that Eip74 activates the enhancer itself prior to treatment and is then depleted of cofactors, a phenomenon called transrepression that is known for hormone signaling pathways (Shlyueva, 2014).

Previous studies showed that hormone receptors bound predominantly to regions that were already accessible prior to treatment and suggested that the chromatin might predetermine hormone-responsive enhancers (Hurtado, 2011; John, 2011). Enhancers were also found that were activated by ecdysone signaling andopen prior to treatment (e.g., a strong enhancer in the Eip75 locus). Some of these open enhancers are already bound by the EcR, which might premark regions to prepare them for fast activation or repress them in the absence of ligand, which is an established function of the EcR. The latter -- 'default repression' -- is another hallmark of TFs and enhancers downstream of signaling pathways, which might ensure reliable regulatory switching. No evidence was found for default repression via the EcR and its motifs, because disruption of EcR motifs in several enhancers did not activate them (Shlyueva, 2014).

The majority of the ecdysone-activated enhancers (>60%) are, however, closed prior to treatment with no detectable DHS-seq signal. The discrepancy between these results and the ones for the ERa and GR above might stem from the fact that not all ERa and GR binding sites determined by ChIP-seq correspond to functional hormone-responsive enhancers . Interestingly, a recent study that considered ERa binding sites that produced eRNAs and were thus likely active (Hah, 2011) concluded that ERa can access and activate enhancers in closed chromatin (Hah, 2013). Together, the current findings that are based on directly assessing enhancer activities caution the interpretation of TF binding sites determined by ChIP: because TFs (and other proteins including GFP) can be frequently crosslinked to open chromatin, the majority of ChIP-seq signals might not correspond to active enhancers. Furthermore, it questions the validity of the frequently used categorization of TF binding sites into enhancers that are regulated positively or negatively based on the flanking genes' transcriptional responses. Even TFs that function exclusively as activators will have binding sites near downregulated genes, such that a repressive function might erroneously be assumed. For example, contrary to prior expectations, only the activating function of Oct4 appears to be required for pluripotency, and this study shows that ecdysone-mediated repression is indirect and independent of EcR binding (Shlyueva, 2014).

In summary, the use of the activity-based enhancer screening method STARR-seq allowed genome-wide identification of functional hormone-responsive enhancers. Combined with the computational dissection of sequence requirements, this approach revealed that the EcR functions together with cell-type- specific partner factors, which are required for enhancer activation. The study also establishes STARR-seq as the method of choice to screen for inducible enhancers downstream of signaling pathways. The combination of STARR-seq with sequence analyses promises to be a useful approach applicable to detect signaling-dependent enhancers and elucidate their sequence characteristics more generally for different signaling pathways and across organisms (Shlyueva, 2014).

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