BIOLOGICAL OVERVIEW

Induction of molting in Drosophila coincides with release from the ring gland of 20-hydroxyecdysone, also known as ecydsone. Prior to each of the larval molts, at pupariation, at pupation and during metamorphosis, hormone is released in carefully timed spurts, coinciding with major morphological transitions. (A description of these stages is give in Developmental origin of adult structures ).

Studies with other insects shows that release of Ecdysone from the ring gland is triggered by the prothoracicotropic hormone, produced by four dorsolateral neurosecretory cells of brain (see Drosophila Prothoracicotropic hormone.

Puffing is the term for changes in polytene chromosomes. The idea that puffing represents gene activity is currently 40 years old. A temporal pattern to puffing in the salivary glands of larval flies is inducible by ecdysone injection. A small number of genes react by puffing within minutes of exposure to ecdysone, and a much larger number (>100) react within hours. It is hypothesized that the time sequence of puffing represents a genetic hierarchy of gene activation. Early puffs are independent of protein synthesis while late puffs require prior protein synthesis (Ashburner, 1990).

Ecdysone receptor is induced at the beginning of the gene activation hierarchy. EcR is induced directly by ecdysone, and provides an autoregulatory loop that increases the level of receptor protein in response to the hormone ligand. EcR exists in three isoforms, each one having an different biological function. Each requires as a partner in heterodimerization the protein Ultraspiracle, the Drosophila homolog of vertebrate RXR proteins. Although ECR can bind ecdysone on its own, binding is greatly stimulated by the addition of USP. Ligand binding stabilizes the ECR-USP heterodimer and increases its affinity for binding to ecdysone response elements in the promoters of genes.

At least five other genes, and probably more than a dozen, are of critical importance to the regulatory hierarchy directed by EcR. E75 is also induced as an early gene, one that codes for another hormone receptor superfamily transcription factor with multiple protein isoforms. Binding sites for EcR exist in the promoter of E75, and EcR is required for the induction of the early response. The E75 response is self delimiting, as transcription is terminated soon after it initiates.

Several other genes act as delayed early genes, including hormone receptor superfamily genes E78B and DHR3, both of which are induced in a delayed fashion after EcR induction. Both require ecdysone-induced protein synthesis for their maximal levels of transcription, and appear to function as monomers to control expression of target genes (Horner, 1995). The delayed timing of E78B and DHR3 induction may allow EcR and E75 to perform regulatory functions before the delayed early genes become active, but the function of these genes is still unknown (Thummel, 1995).

During a second wave of puffing 4 to 6 hours after puparium formation, ßFTZ-F1 is induced as a mid-prepupal gene. FTZ-F1 maps to the 75CD mid-prepupal puff. It has been shown to interact with elements of the alcohol dehydrogenase gene and has also been implicated as an activator of fushi tarazu. ßFTZ-F1 is another complex gene with multiple isoforms; antibodies to ßFTZ-F1 detect binding to 166 loci in late prepupal salivary gland polytene chromosomes, 51 of which represent ecdysone-regulated puffs. Of 33 puffs that show increased activity after the peak of the 75CD puff, 17 show reproducible staining for ßFTZ-F1 (Lavorgna, 1993).

Experiments with cultured larval salivary glands have demonstrated that ßFTZ-F1 transcription is negatively regulated by ecdysone. In the absence of ecdysone, ßFTZ-F1 is induced. Repression is overcome as the levels of both ecdysone and EcR decrease during early-prepupal development. Thus ßFTZ-F1, through its interaction with EcR, provides a molecular mechanism for stage-specific responses to steroid hormones (Woodard, 1994).

In late prepupae, the midprepupal puffs regress and the early puffs are reinduced. In addition, a few stage-specific early puffs, typified by E93, are directly induced by ecdysone in late prepupae, but not in late larvae. The early puffs cannot be induced by ecdysone in early-prepupal salivary glands. Rather, a preceding period of protein synthesis and low ecdysone concentration is required before these puffs become competent to respond to hormone. It is suggested that one or more proteins encoded by the mid-prepupal puff genes provide the compentence for the early puffs to be induced by the prepupal ecdysone pulse; FTZ-F1 is a good candidate for this required gene (Woodard, 1994). Negative regulation is required in molting, as much as positive effects. For example, DHR78, an orphan nuclear receptor expressed throughout the early stages of metamorphosis, cannot heterodimerize with either ECR or USP but can bind to an Ecdysone receptor response element of a downstream gene in the hierarchy inhibiting the ability of ECR and USP to induce transcription (Zelhof, 1995).

In conclusion, each of these gene dyads and triads; EcR and USP, E75A, E78B and DHR3, ßFtz-F1, DRH78 and E93, is required in a sequential genetic hierarchy for the the timing of metamorphosis and the induction and repression of genes required for the differentiation process (Thummel, 1995). The complexity of the insect molting hierarchy serves as a warning for a generation of scientists who would unravel the hierarchies of mammalian development.

A role for juvenile hormone in the prepupal development of Drosophila melanogaster

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met²⁷ larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells 'misinterpreting' the ecdysteroid peaks that drive metamorphosis (Riddiford, 2010).

Insect molting and metamorphosis are governed primarily by ecdysone (used in the generic sense) and juvenile hormone (JH), with ecdysone causing molting and JH preventing metamorphosis. Juvenile hormone has a classic 'status quo' action in preventing the program-switching action of ecdysone during larval molts and in maintaining the developmental arrest of imaginal primordia during the intermolt periods. Its effects at the outset of metamorphosis, though, are more complex. Studies mainly on Lepidoptera show that for selected tissues JH needs to be present to allow them to undergo pupal differentiation, rather than undertaking a precocious adult differentiation (Riddiford, 2010).

The mechanism through which JH maintains the status quo and directs early development at metamorphosis is still poorly understood. Whether JH has one or multiple receptors, and the nature of these receptors, is still controversial. The best candidate for a receptor is the product of the Methoprene-tolerant (Met) gene, a PAS domain protein that was originally isolated in Drosophila melanogaster. In vitro transcribed and translated Met protein has been shown to bind JH with high affinity, and RNAi knock-down experiments in Tribolium castaneum show that Met is essential for mediating the status quo action of JH in this beetle (Riddiford, 2010).

In D. melanogaster, JH is thought to have no role in the onset of metamorphosis, since exogenous JH only delays but does not prevent pupariation. Although it has no apparent effect on the development of the imaginal discs, JH prevents normal adult development of the abdominal integument when given at pupariation. Internally, JH at this time affects normal reorganization of the central nervous system and development of the thoracic musculature. These effects of JH on metamorphosis do not occur in Met mutants, unless at least 100 times the dose is given. The Met²⁷-null mutants proceed through larval development and metamorphosis apparently normally. However, if in addition, RNAi is used to suppress expression of Germ-Cell Expressed (Gce), a related bHLH protein with a high similarity to Met that heterodimerizes with it, Met-null mutants die as pharate adults. In the Met-deficient mutant, the adult eye shows a few (<12) defective ommatidia in the posterior region. Also, the females mature fewer eggs at a slower rate than do wild-type females, indicating that Met is also important for JH effects in egg maturation (Riddiford, 2010).

This study genetically allatectomized Drosophila larvae by targeting expression of a cell death gene to the corpora allata (CA), the gland that produces JH. These larvae form smaller puparia and showed precocious maturation of the visual system, but die around head eversion (Riddiford, 2010).

Although a number of studies have reported the effects of applying exogenous JH or JH mimics to Drosophila, there are only two very recent studies of the effects of manipulating endogenous JH on larval growth and metamorphosis, both of which appeared while this paper was under review. JH is normally present in the early larval instars, declines substantially during the last (third) larval stage and then returns transiently around the time of pupariation. The allatectomized (CAX) larvae undergo the expected two larval molts, but because sometimes the remains of degenerating CA cells are seen at the start of the last larval stage, nothing can be concluded about the requirements of JH for these larval molts. Recently, Jones (2010) using 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) RNAi to depress the level of JH and its farnesoid precursors in early larvae, showed that the larvae mainly die during the molt to the third instar, indicating that JH may be required for that molt (Riddiford, 2010).

The destruction of the CA by the third instar allowed examination of the role of JH during the last instar and early metamorphosis. The finding that these larvae were smaller than their CyO, UAS-grim siblings at pupariation could be explained by either the loss of JH or by the loss of the salivary glands, since these glands are also destroyed. Because dietary JH in the final instar rescued these larvae to normal size, the lack of the CA, rather than the lack of the salivary glands, is the cause of their reduced growth. Preliminary studies show that CAX larvae grow more slowly in the third instar, but the underlying basis for this retardation is not yet understood. Similarly, allatectomized third instar larvae display premature apoptosis of the fat body and downregulation of several enzymes involved in energy metabolism at the onset of wandering. These fat body effects could underlie the reduced larval growth seen in CAX larvae (Riddiford, 2010).

A major effect of the removal of JH was on the timing of events during the prepupal period. Studies on the wild silkmoth Hyalophora cecropia first showed that removal of the CA in the last larval stage resulted in the formation of a pupa with adult characteristics. Other moths, like Manduca sexta, showed more subtle responses to allatectomy, with premature adult differentiation most evident in the patterned region of the compound eye, posterior to the morphogenetic furrow. Subsequent studies on a variety of tissues in Manduca showed that the eye, the optic lobe and the ventral diaphragm each had a prolonged period of proliferation that extended from the prepupal period through early adult differentiation. This proliferation is maintained by α-ecdysone or low levels of 20-hydroxyecdysone (20E), but is terminated by high levels of 20E, which induces differentiation. These tissues are exposed to differentiation-inducing titers of 20E that occur during the larval-pupal transition early in their growth, but studies on the ventral diaphragm showed that JH 'protects' them from these high 20E levels, allowing them to continue proliferating. Removal of JH results in these tissues undergoing premature termination of tissue growth and precocious adult differentiation (Riddiford, 2010).

The response of Drosophila larvae to the loss of JH is in line with the effects seen in Manduca, and is also most evident in the developing visual system. In normal individuals, the appearance of EcR-B1 in the optic lobe and the termination of proliferation in the outer proliferation zone coincide with the ecdysteroid peak at head eversion and become more pronounced at 18 hours APF with the rise of ecdysteroid for adult differentiation. The separation of the R7 and R8 growth cones also begins about this latter time. The only one of these tissues that has been directly tested in vitro for 20E sensitivity is the optic lobe and in this case high levels of 20E do indeed suppress proliferation. It is assumed that the other processes also respond to the changing ecdysteroid levels. The lack of JH results in a heterochronic advance of these events by 10 to 12 hours, consistent with the tissues now responding to the earlier ecdysteroid peak that causes pupariation. Although the removal of JH advances these processes, it was found that the application of JH mimics delays them. Consequently, in selective tissues in Drosophila, JH acts to direct the nature of tissue responses to ecdysone (Riddiford, 2010).

The removal of JH or of one its receptors, Met, has a mixed effect on the developing visual system. No effect on proliferation or inductive events was seen in the eye disc itself, in that in CAX animals the morphogenetic furrow continues to move and similar rows of ommatidia have sent R8 axons into the medulla by 6 hours APF, as compared with controls. Likewise, in Met²⁷ individuals there is only a slight advance (about 2 hours) in the schedule of lamina interneuron ingrowth into the medulla. However, for some of the cellular and molecular events, like the appearance of EcR-B1 and the separation of R7 from R8, there is a 10- to 18-hour advance in their occurrence. Hence, the lack of JH or of its receptor Met causes a heterochronic shift within the developing visual system with some differentiation responses being advanced relative to the normal schedule of neuronal birth and axon ingrowth. At least in the case of the photoreceptors, the effect of Met removal is largely cell autonomous, with the reduction of Met function in just those cells being sufficient to cause the precocious appearance of EcR-B1 and the early separation of R7 from R8. By contrast, the reduction of Met in lamina interneurons allowed these cells to precociously express EcR-B1 but did not affect the behavior of the R7 and R8 growth cones. This suggests that the separation of R7 and R8 is an active response of the photoreceptors, which is likely to be caused by the rising ecdysteroid titer driving adult differentiation. Although the lack of JH or Met function at the outset of metamorphosis results in the cell-autonomous expression of EcR-B1 in the photoreceptors, misexpression experiments show that the appearance of this receptor alone is not sufficient to bring about the early separation of R7 and R8. Therefore, although the upregulation of EcR-B1 is a prominent response to rising ecdysteroid titers, it is not the key change responsible for the repositioning of the receptor terminals (Riddiford, 2010).

As it is viable, the Met mutant allowed the final results of the mistiming of development in the optic lobes to be seen. No permanent effect was seen of the early separation of the R7 and R8 growth cones on the final anatomy of these projections in the medulla, or on the structure of the later neuropil. However, the lobula was grossly distorted and the normal layering of dendritic arbors disrupted. This aberrant morphogenesis also starts early, being already evident by 12 hours APF. The cellular basis for the lobula distortion, however, is not yet known (Riddiford, 2010).

Heterochronic shifts in the timing of development that extend beyond the visual system are likely to be the cause of the lethality seen in the CAX puparia. Puparia appear normal through the first 6 to 7 hours after pupariation but then abruptly undergo tissue collapse. In normal flies, the early part of metamorphosis is accomplished by a complicated replacement of histolyzing larval tissues by the growing adult tissues. Diverse tissues show individualized times of histolysis that are tied to the ecdysteroid titer. For instance, the larval midgut cells degenerate in response to the pupariation peak of ecdysone, whereas the larval salivary gland degeneration is triggered by the small rise of ecdysteroid at the end of the prepupal period. It is suspected that without JH, some of the histolysis events are mistimed, leading to the rapid death of the prepupa. It has been shown in CAX larvae that the fat body undergoes precocious programmed cell death beginning in the third larval instar. Interestingly, this lethal effect was not seen in animals in which Aug21-GAL4 drove RNAi for JH acid O-methyltransferase, the enzyme that converts JH acid to JH, in the CA (Niwa, 2008). Whether this indicates that JH acid plays a role in prepupal development or merely reflects the incomplete loss of JH in these animals is unknown (Riddiford, 2010).

All these effects of allatectomy can be rescued by JH either fed during the third instar or applied at the time of early wandering, but not at pupariation. A decline of JH III occurs in the third instar; this is followed by a peak of JH during late wandering. When JH begins to rise is unknown, as measurements were made every 24 hours. Presumably it is the lack of this JH during wandering when the ecdysteroid titer is rising and peaking that leads to the optic lobe anomalies and the premature histolysis (Riddiford, 2010).

The finding that the Met²⁷ null mutant has the same defects in optic lobe development as are found in CAX prepupae strongly suggests that JH is acting via the Met pathway in controlling the timing of some events in the optic lobe. Accordingly, JHM treatment cannot suppress most of the premature development seen in prepupae lacking Met. However, a major difference between the CAX animals and the Met²⁷ mutants is that the CAX prepupae died before head eversion, whereas the Met²⁷ animals are viable. This difference is also seen in the precocious cell death of the fat body caused by allatectomy, which does not occur in the Met null mutant even in the presence of gce RNAi. Instead precocious cell death of the fat body was seen when Met was overexpressed in that tissue and the death could be suppressed by exogenous methoprene (a JH mimic). This latter finding suggests that JH would act in this case to suppress Met-mediated cell death. This idea was tested by seeing whether the removal of Met would protect the prepupa from the death caused by early allatectomy. When Met²⁷; Aug21-GAL4>UAS-GFP/CyO females were crossed with UAS-grim males, 44% eclosed, all showing the CyO phenotype. The remainder died at head eversion, and should have been half CAX, Met-heterozygous females and half CAX, Met-null males. Another group was separated by sex prior to pupariation. Forty-nine percent of the females and 48% of the males died at head eversion. All of the adults that emerged were CyO, showing that all the CAX prepupae died regardless of whether or not they were lacking Met function (Riddiford, 2010).

These results together with the findings that JHM treatment of the Met²⁷ mutant gave a partial rescue of the premature separation of R7 and R8, and of the decreased proliferation in the inner proliferation zone, indicate that there may be more than one receptor for JH. Thus, JH might act through multiple pathways. A major pathway involves Met, but Gce or some other mediator may serve as an alternate pathway in some tissues. A similar protective role of JH at pupation mediated by Met is found in Tribolium; injection of Met RNAi into either fourth instar larvae or final instar larvae caused the precocious appearance of adult eyes, adult antennae and other features in the resulting pupae (Riddiford, 2010).

These studies show that JH has an endogenous function in regulating Drosophila metamorphosis, a specific example being in orchestrating the timing of differentiation events in the developing visual system. These effects of JH are primarily mediated through the Met pathway. JH also is necessary for normal larval growth and has another, as yet undefined, crucial role in prepupal development that prevents death at head eversion. The latter effect is not mediated through Met, indicating that JH might act through multiple pathways (Riddiford, 2010).

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; Phago^less 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).

Steroid signaling promotes stem cell maintenance in the Drosophila testis

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). This study shows that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which has been shown to maintain CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance (Li, 2014).

This work shows that the steroid hormone 20E plays an important role in maintaining stem cells in theDrosophila testis: 20E, receptors of ecdysone signaling, and downstream targets are required directly in CySCs for their maintenance. When ecdysone signaling is lost in CySCs, GSCs are also lost, but it is unclear if their maintenance requires an ecdysone-dependent or independent signal from the CySCs. The requirement for EcR in the testis is isoform-specific: expression of EcR-B2 in the CySC lineage is sufficient to rescue loss of GSCs and CySCs and increased cell death in EcR mutant testes, suggesting that there might be a temporal and spatial control of ecdysone signaling in the adult testis. In addition, evidence is provided that ecdysone signaling, as in the ovary, is able to interact with an intrinsic chromatin-remodeling factor, Nurf301, to promote stem cell maintenance. Therefore, these studies have revealed a novel role for ecdysone signaling in Drosophila male reproduction (Li, 2014).

Although ecdysone signaling is required in both ovaries and testes for stem cell maintenance, the responses in each tissue are likely to be sex-specific. In the ovary, 20E controls GSCs directly, by modulating their proliferation and self-renewal, and it acts predominantly through the downstream target gene E74. In contrast, male GSCs require ecdysone signaling only indirectly: ecdysone signaling was found to be required in the CySC lineage to maintain both CySCs and GSCs. In a previous study, RNAi-mediated knockdown of EcR, usp or E75 in the CySC lineage did not result in a significant loss of GSCs; however, the number of CySCs was not determined, and the phenotype was examined after 4 or 8 days, not 14 days as in this study. It is suspected that the earlier time points used in that study may not have allowed enough time for a significant number of GSCs to be lost (Li, 2014).

During development, 20E is produced in the prothoracic gland (PG) and further metabolized to 20E in target tissues, but the PG does not persist into adulthood. In adult female Drosophila, the ovary is a source of 20E. In contrast, the identification of steroidogenic tissues in adult male Drosophila remains the subject of active investigation. The level of 20E in adult males is significantly lower than in adult females, but it can be detected in the testis. Furthermore, RNA-seq data show that shade, which encodes the enzyme that metabolizes the prohomone ecdysone to 20E, is expressed in the adult testis, suggesting that the adult testis may produce 20E. However, the sources of 20E production in adult Drosophila males remain to be determined experimentally (Li, 2014).

20E, like other systemic hormones, can have tissue-specific effects or differential effects on the same cell type as development proceeds. These differences are mediated at least in part by the particular downstream target genes that are activated in each case. For example, in female 3^rd instar larval ovaries, ecdysone signaling upregulates br expression to induce niche formation and PGC differentiation, but br is not required for GSC maintenance in the adult ovary; instead, E74 plays this role. Similarly, br is required for the establishment of intestinal stem cells (ISCs) in the larval and pupal stages but not for ISC function in adults. This study shows that ecdysone signaling in the adult testis is mediated by different target genes than in the ovary: E74, but not E75 or br, regulate stem cell function in the ovary, whereas E75 and ftz-f1 are important for stem cell maintenance in the testis. Since E75 is itself a nuclear hormone receptor that responds to the second messenger nitric oxide, it will be interesting to know whether E75's partner DHR3 also plays a role in CySCs. An intriguing question for future studies will be how different ecdysone target genes interact with the various signaling pathways that maintain stem cells in the ovary or testis (Li, 2014).

Since 20E levels can actively respond to physiological changes induced by environmental cues, it is possible that the effect of 20E on testis stem cell maintenance might reflect changes in diet, stress, or other environmental cues. For example, in Aedes aegypti, ecdysteroid production in the ovary is stimulated by blood feeding and this is an insulin-dependent process. In Drosophila, ecdysone signaling is known to interact with the insulin pathway in a complex way. Ovaries from females with hypomorphic mutations in the insulin-like receptor have reduced levels of 20E. Furthermore, ecdysone signaling can directly inhibit insulin signaling and control larval growth in the fat body. Thus, ecdysone signaling may interact with insulin signaling during testis stem cell maintenance. Previously, it was shown that GSCs in the ovary and testis can respond to diet through insulin signaling, which is required to promote stem cell maintenance in both sexes. It is possible that diet can affect 20E levels and thus regulate stem cell maintenance. In addition to diet, stress can also affect 20E levels, as is the case in Drosophila virilis, where 20E levels increase significantly under high temperature stress. A similar effect has been found in mammals, where the steroid hormone cortisol is released in response to psychological stressor. Finally, 20E levels are also influenced by mating. In Anopheles gambiae, males transfer 20E to blood-fed females during copulation, which is important for egg production. In female Drosophila, whole body ecdysteroid levels also increase after mating. Studying the roles of hormonal signaling in mediating stem cell responses to stress and other environmental cues will be an exciting topic for future studies. From this work it is now clear that, as in mammals, steroid signaling plays critical roles in adult stem cell function during both male and female gametogenesis (Li, 2014).

Modulators of hormonal response regulate temporal fate specification in the Drosophila brain

How a progenitor sequentially produces neurons of different fates and the impact of extrinsic signals conveying information about developmental progress or environmental conditions on this process represent key, but elusive questions. Each of the four progenitors of the Drosophila mushroom body (MB) sequentially gives rise to the MB neuron subtypes. The temporal fate determination pattern of MB neurons can be influenced by extrinsic cues, conveyed by the steroid hormone ecdysone. This study shows that the activation of Transforming Growth Factor-beta (TGF-beta) signalling via glial-derived Myoglianin regulates the fate transition between the early-born alpha'beta' and the pioneer alphabeta MB neurons by promoting the expression of the ecdysone receptor β1 isoform (EcR-β1). While TGF-beta signalling is required in MB neuronal progenitors to promote the expression of EcR-β1, ecdysone signalling acts postmitotically to consolidate the alpha'beta' MB fate. Indeed, it is proposed that if these signalling cascades are impaired alpha'beta' neurons lose their fate and convert to pioneer alphabeta. Conversely, an intrinsic signal conducted by the zinc finger transcription factor Kruppel-homolog 1 (Kr-h1) antagonises TGF-beta signalling and acts as negative regulator of the response mediated by ecdysone in promoting alpha'beta' MB neuron fate consolidation. Taken together, the consolidation of alpha'beta' MB neuron fate requires the response of progenitors to local signalling to enable postmitotic neurons to sense a systemic signal (Marchetti, 2019).

This study reveals a fundamental role for Myo-mediated TGF-β signalling in regulating fate specification of MB neurons. This signalling is initiated in the neuronal progenitors and it is proposed that it is necessary to consolidate the identity of newly born neurons by enabling them to sense and integrate the ecdysone hormonal signal. As modulator of this consolidation fate program, the factor Kr-h1 negatively regulates ecdysone signalling response and antagonises the TGF-β pathway (Marchetti, 2019).

Evidence derived from vertebrate models indicates that the temporal competence of neuronal precursors to generate different neuronal subtypes is governed by the combination of cell-intrinsic programs and extrinsic cues. In contrast, fate determination in the Drosophila nervous system appeared to be mainly determined by intrinsic cascades. Only recently, first reports started indicating that extrinsic factors can modulate fate decisions in the nervous system of the fly. Thus, fate decisions in the fly nervous system might follow principles that are more relatable to the ones utilised in vertebrate lineages than previously expected. Along these lines, the current data revealed a central role of TGF-β signalling in temporal fate specification during MB development. In the rodent hindbrain, midbrain and spinal cord, TGF-β signalling constrains the neural progenitor potency to promote fate transition from early to late born cell types, acting as a temporal switch signal regulating the expression of intrinsic identity factors in young progenitors. These similarities suggest that TGF-β might represent an evolutionary conserved extrinsic signal modulating temporal fate specification (Marchetti, 2019).

The present data suggest that TGF-β signalling links the temporal neuronal fate program to developmental progression. Re-examination of the EcR-β1 expression in dSmad21 mutant MB clones at late larval stages revealed a 12 hours delay in the onset of EcR-β1 expression leading to inability of MB neurons to respond to the prepupal ecdysone peak. Thus, TGF-β signalling might help to synchronize the production of distinct MB neuron subtypes coordinating diverse developmental programs. Accordingly, this study found that the glial Myo ligand mediates the TGF-β-dependent MB fate transition. Given that the prepupal ecdsyone peak is triggered after the larva reaches the critical weight point, it was hypothesise that glia serve as nutrition sensors in the brain during larval development and could be coordinating developmental timing of the fate specification program (Marchetti, 2019).

Although α'β' neurons are born during the larval stage, based on their immature dendrites and axons, and on the absence of functional response in appetitive olfactory learning behaviour, it appears that they are not fully differentiated at the end of larval life. Therefore, the initial state of these immature α'β' neurons could be labile. Their immature neurite trajectories might possess a certain degree of morphological plasticity, since at early pupal stages the axonal lobes are primarily made of α'β' axons, after γ axons have completely pruned. Indeed, the data provide strong support for the presence of an active consolidation signal required to maintain α'β' fate at adult stage. In fact, after impairment of TGF-β signalling, neurons born in the time window corresponding to the production phase of α'β' displayed the expected axonal pattern for α'β' neurons and expressed an α'β' marker before metamorphosis. Taken these data together, the alternative hypothesis that TGF-β signalling could be involved in the initial specification of α'β' MB neurons at mid-third instar appears much more unlikely. Notably, studies on fate specification in vertebrate systems have described a postmitotic fate consolidation event for developing motor and cortical neurons. In particular, the homeobox gene HB9 has an essential function in maintaining the fate of the motor neurons by actively suppressing the alternative V5 interneuron genetic program. Indeed, mice lacking HB9 function showed a normal number of motor neurons that acquired, though, molecular features of V5 interneurons. Interestingly, in absence of HB9 motor neurons are initially specified and they retain their characteristic axonal projection. Similarly, the expression of the retinoic acid receptor (RAR) is required to maintain the fate of layer V-III cortical neurons, and when the expression of RAR is abolished these neurons acquire the identity of layer II cortical neurons. These similarities in fate consolidation programs might reflect a common strategy in both invertebrates and vertebrates to first specify and then refine neuronal fate, according to the appropriate context (Marchetti, 2019).

Recently, RNA profiling analysis of MB neurons at different developmental time points uncovered a complex feedback regulation network that governs EcR expression. This combination of positive as well as negative feedback loops is required to coordinate EcR expression levels and its temporal regulation during brain development. FISH analysis suggested that TGF-β signalling promotes the transcription of the EcR-β1 gene in MB neurons at late wandering larval stage. Although detectable EcR-β1 protein is restricted to postmitotic MB neurons, genetic data revealed that TGF-β signalling is necessary in the MB progenitors to allow the expression of EcR-β1. This evidence raises the possibility that TGF-β signalling promotes the transcription of EcR gene in neuronal progenitors and potentially post-transcriptional mechanisms are involved to narrow down the translation of the EcR-β1 receptor only postmitotically. However, the data are against this hypothesis, since expression of EcR-β1 specifically in MB progenitors did not rescue the TGF-β signalling-dependent fate defects. Moreover, given that TGF-β signalling is required to consolidate the fate of the larval-born α'β' neurons at the end of larval stage, suggests that the TGF-β pathway regulates a consolidation fate process independently of cell division. In this scenario, the expression of EcR-β1 in the newly born neurons could be promoted via a cell-to-cell communication signalling cascade initiated in neuronal progenitors by the activity of TGF-β signalling. Examples of this type of signal transmission are represented by the juxtacrine signalling mediated by Notch, Semaphorin or Ephrin pathways. In particular, the intercellular interaction between Notch and its ligand Delta in neighbouring cells is fundamental to direct cell fate decisions (Marchetti, 2019).

In addition to an upstream regulation of ecdysone signalling, this study uncovered the intrinsic factor Kr-h1 as a downstream modulator of the ecdysone-dependent fate consolidation program. Interestingly, the transition from larval stage to metamorphosis is regulated by the balance of the two major hormones, the juvenile hormone (JH) and ecdysone. JH prevents metamorphosis by the induction of the transcription factor Kr-h1 within the ring gland, which in turn suppresses the up-regulation of the ecdysone-dependent metamorphic genes E93 and Broad Complex. The TGF-β/Activin pathway contributes to decreasing Kr-h1 expression via E93 allowing the beginning of metamorphosis. Along these lines, the antagonism between ecdysone and JH through Kr-h1 could potentially regulate the MB temporal fate cascade at the onset of metamorphosis (Marchetti, 2019).

In conclusion, this work shed light on the intrinsic and extrinsic mechanisms regulating the consolidation of the terminal fate. Understanding these processes will help gain insights into their dysregulation in neurodevelopmental disorders and into their role in stem cell reprogramming (Marchetti, 2019).

Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation

The activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. This study shows that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. The Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) was identified as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. The steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. These findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, these findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation (Christensen, 2020).

The activation of a neuroendocrine signaling cascade triggers maturation onset in most animals. This activation is associated with body-size gating to ensure the fitness of the reproductive adult. In insects, attainment of 'critical weight' during the last larval instar is the main such checkpoint gating the transition to adulthood. After this checkpoint, a larva becomes committed to maturing on a fixed schedule irrespective of further nutrition. Thus, critical weight likely reflects energy stores sufficient to survive the non-feeding maturation process (metamorphosis) and obtain a final adult body size that maximizes fitness. Nutritional status is likewise a main factor permitting the entry into maturation in mammals. In humans, body weight correlates with the timing of menarche, which led to the use of the term 'critical weight' for the onset of reproductive cycles in humans. Obese children enter puberty earlier than height-matched non-obese children, and malnutrition and lack of body fat can lead to delayed puberty. These observations suggest that the maturation gate reflects not body size per se but rather the amount of body fat, and thus that the neuroendocrine system controlling the timing of this process somehow assesses nutritional and energetic stores. Interestingly, the adipokine leptin regulates pubertal maturation in mammals. Circulating leptin levels correlate with adiposity, and leptin-deficient humans and mice fail to undergo puberty. Leptin may therefore communicate body-fat levels to the neuroendocrine system controlling puberty, which could explain the link between childhood obesity and early onset of puberty. In insect larvae, the fat body is the main nutrient-storage and -sensing organ, releasing numerous nutrient-dependent insulin-regulating hormones. Insulin is a stimulator of ecdysone production, thus coupling adipose-tissue nutrient sensing to the neuroendocrine maturation axis in Drosophila. Among the insulinotropic adipokines is Unpaired 2 (Upd2), which is structurally and functionally similar to human leptin. Upd2 acts through the JAK/STAT receptor Domeless (Dome) in GABAergic neurons that regulate insulin secretion from the insulin-producing cells (IPCs) in the brain, which are the primary source of circulating insulin. Thus, related adiposity hormones that signal nutrition and energy storage influence the neuroendocrine events that lead to the onset of maturation in divergent systems (Christensen, 2020).

Mammalian GnRH-producing neurons regulate the timing of puberty onset, and these cells are activated by the neuropeptide KISS1. The PTTHn, activated by the KISS1 ortholog AstA and its receptor AstA-R1 (Deveci, 2019), serve this function in Drosophila. This suggests conservation of the overall neuroendocrine architecture of the maturation-initiation system. AstA is regulated by nutritional intake, providing another potential link between energy status and maturation onset. Furthermore, PTTHn-specific knockdown of Insulin receptor (InR) or dome, encoding the Upd2 receptor, produced size phenotypes in the screen carried out in this study, suggesting that the PTTHn integrate systemic nutrition-regulated signals and may also receive input via insulin from the IPCs themselves. Because PTTH controls developmental timing, and insulin is the main growth-regulatory factor, these results suggest that Upd2 may link growth and maturation by coordinating the activity of both the IPCs and the PTTHn. Knockdown of the amino-acid transporters Polyphemus and Minidiscs also induced strong growth effects in the screen, suggesting that the PTTHn may also sense nutrient status autonomously; in the IPCs, Minidiscs is required for inducing insulin secretion after intake of the amino acid leucine (Christensen, 2020).

This raises the key question of how these nutritional cues lead to the surge mode of GnRH/PTTH release that initiates maturation. The current findings suggest that ecdysone feedback, via EcR in the PTTHn, is the mechanism that induces the PTTH and ecdysone surge towards the end of larval development. This is further reinforced by EcR-mediated positive feedback on ecdysone production in the PG. It is proposed that the triggering event that begins the feedback cycle is a small nutrient-dependent ecdysone peak early in the L3 stage. Nutritional signaling via insulin acts directly on the PG and is required for ecdysone production pre-critical weight but not post-critical weight. Furthermore, PTTH secretion is also controlled by nutrition and is required for normal attainment of critical weight, suggesting that PTTH acts together with insulin before attainment of critical weight to generate a small nutrient-dependent rise in ecdysone production at the beginning of L3. This small ecdysone peak upregulates Ptth via EcR and, under this scenario, corresponds to critical weight, which occurs ~10 h after the L2-L3 transition. Thus, when ecdysone reaches the threshold corresponding to critical-weight attainment, it generates an irreversible, self-sustaining feedback activation of the neuroendocrine system by promoting the PTTH surge that triggers the maturation-inducing ecdysone pulse towards the end of L3. This model is supported by findings showing that a small nutrient-sensitive ecdysone peak early in L3 does indeed signal critical weight (Christensen, 2020).

The main feature of this model is ecdysone feedback onto the PTTHn via a mechanism requiring EcR in these cells. EcR/Usp may regulate Ptth expression by direct binding to the Ptth enhancer or through downstream target transcription factors regulated by this complex. Many transcription factors are known to be targets of EcR, and EcR may indirectly regulate Ptth expression by altering the expression of one or more of these. Indeed, RNAi against certain known EcR-induced transcription factors, such as Hr39, Hr3 and ftz-f1, produced phenotypes in the screen, consistent with a possible role in Ptth regulation. Hr3 and Ftz-F1 are also known to participate in ecdysone regulation in the PG, as is another nuclear receptor, Knirps, which was also identified in the screen as a potential regulator of PTTH. Clarifying the precise mechanism by which EcR controls Ptth expression will be an interesting topic for future investigation (Christensen, 2020).

Early maturation is associated with smaller adult size in both flies and humans, as this event limits the juvenile growth period. The prevalence of precocious puberty has been linked with the increasing rates of childhood obesity; however, the mechanisms that gate GnRH secretion at the time of puberty are poorly understood. The mammalian HPG axis controlling the onset of puberty is regulated by feedback control in which steroid hormones act to regulate the GnRH-expressing neurons, but whether these neurons themselves are direct steroid targets is still debated. However, the KISS1-expressing neuronal population has also emerged as a possible link between sex steroids and the GnRH neurons (Christensen, 2020).

Many studies in Drosophila and other insects have explored the neuroendocrine PTTH-PG-ecdysone axis. The existence of feedback control between ecdysone and PTTH has been hypothesized for decades and is supported by studies of PTTH in other insects, especially in lepidopterans, in which hemolymph titers of PTTH and ecdysone are clearly correlated during the last larval instar. A PTTH surge immediately precedes a rise in ecdysone levels, and gradual increases in ecdysone levels appear to reinforce the peak levels of circulating PTTH, suggesting that ecdysone might positively influence PTTH release. This is supported by findings that injection of ecdysone before an endogenous PTTH peak induces a premature rise in PTTH, whereas injection of ecdysteroid-22-oxidase, a potent enzymatic inactivator of ecdysteroids, inhibits this rise. This study has demonstrated an EcR-dependent positive-feedback mechanism, operating specifically within the PTTHn, that regulates the transcription of Ptth. The findings show a mechanism by which steroid-mediated feedback signaling triggers the PTTH surge at the onset of metamorphosis, suggesting that feedback control is an evolutionarily conserved regulator of the neuroendocrine signaling that initiates the onset of maturation (Christensen, 2020).

Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases. This study reports that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size (Ahmed, 2020).

Steroidal sex hormones including oestrogen, progesterone and testosterone regulate the growth and physiology of reproductive organs during puberty, the oestrus cycle and pregnancy. Consequently, these hormones also promote tumorigenesis in the breast, uterus and prostate. Although sex-specific differences in physiology and disease predisposition extend to nearly all organs, the functions of sex-specific steroids in non-sex organs remain relatively poorly explored and controversial. Drosophila uses one major steroid hormone, 20-hydroxy-ecdysone (ecdysone, also known as 20HE) and its derivatives. Similar to vertebrate steroids, 20HE is synthesized by cytochrome P450 enzymes from cholesterol. The ecdysone receptor comprises a ligand-binding EcR subunit and a DNA-binding Usp subunit-orthologues of human farnesoid X and liver X receptors (FXR and LXR) and retinoid X receptor (RXR), respectively. In juvenile insects, 20HE regulates developmental transitions including moulting, metamorphosis and sexual maturation. In adult Drosophila, 20HE is made by the ovaries after mating, resulting in higher levels in females than in males. It acts in the adult nervous and reproductive systems and affects metabolism and lifespan, but a role in the gut has not been described (Ahmed, 2020).

Drosophila intestinal stem cells (ISCs) are more proliferative in females than in males, and females are more prone to age-dependent gut dysplasia and intestinal tumours. These sex-specific traits could be due to ISC-autonomous and/or systemic factors. Consistent with the former, stress-dependent ISC divisions, which are more frequent in females than in males, are reduced if the ISCs are masculinized by repressing the sex-determination genes sxl or tra2. Mated females support more ISC division than virgin flies, which suggests hormonal influences. Because mated females have higher titres of ecdysteroid than virgins or males, tests were performed to see whether 20HE might affect ISC proliferation. Indeed, feeding virgin females 5 mM 20HE strongly induced ISC divisions. This effect was independent of ISC sex identity, and also occurred in mated females and males. Using reporters of receptor activity, it was confirmed that exogenous 20HE promotes EcR-Usp signalling in midgut ISCs, transient progenitors known as enteroblasts (EBs) and differentiated absorptive enterocytes (ECs) (Ahmed, 2020).

Unlike stress caused by detergents, 20HE treatment induced two successive waves of ISC division. Using RNA interference (RNAi) under the control of conditional cell-type-specific Gal4 drivers, it was found that the first wave (at 6 h after 20HE feeding) required EcR only in ISCs, but that later divisions (at 16 h) also depended partially on EcR in EBs. Neither wave of division required EcR in ECs, enteroendocrine or neural cells. Isoform-specific tests revealed that EcR-A was much more important than EcR-B for the 20HE-induced division of ISCs. 20HE-induced divisions were reversible , which suggests a lack of toxicity. EcR activity was not induced by enteric infection, and EcR was dispensable for infection-induced gut regeneration, which indicates a distinct role for EcR in the gut. Loss of Usp, however, did block infection-induced ISC divisions, which suggests that Usp has EcR-independent functions (Ahmed, 2020).

It was next asked whether ISC activation by 20HE involves the Upd-Jak-Stat or Egfr-ERK signalling pathways, which are known to activate ISCs after stress. Six hours of 20HE feeding induced the Egfr ligands spi and krn and their activating protease rho, but not the upd2 or upd3 cytokines or Stat signalling. Exposure to 20HE for 16 h, however, moderately induced upd2, upd3 and Stat activity. The induction of upd2, upd3 and rho required EcR in ISCs and EBs (that is, 'progenitors'), although not in ECs. The Egfr effector ERK was also mildly activated by 16 h of 20HE exposure, mostly in progenitors but occasionally in ECs. ERK activation required upd2, which suggests a signalling relay. Notably, the induction of all of these targets (upd2, upd3, Socs36E, rho, spi and krn) by 20HE was suppressed by blocking ISC mitoses with RNAi molecules that target string (also known as stg or cdc25) or Egfr. This suggests that the observed increases in Jak-Stat and Egfr-ERK signalling are responses to epithelial stress from the early ISC divisions. In further tests, it was found that Upd2 from EBs and ECs contributed strongly to ISC divisions 16 h after 20HE feeding, but only weakly to the early divisions at 6 h. Egfr and Rho, however, were always required. It is concluded that ISC divisions are initially activated ISC-autonomously via EcR, and require Egfr and Rho, whereas later divisions depend in part on cytokines produced by EBs and ECs, perhaps in response to stress from the first mitoses. The relationship of EcR to Egfr signalling warrants further investigation (Ahmed, 2020).

Because mated females produce more ecdysone than virgins or males, whether 20HE might account for sex-specific differences in the gut was tested. Consistent with this, long-term exposure of males to 20HE phenocopied the female condition, increasing ISC mitoses, stress responsiveness, epithelial turnover and midgut size. Genetically feminizing the male ISCs did not give these effect, which suggests that 20HE acts independently of genetic sex determination. Forced expression of the ISC mitogen13 sSpi also failed to enlarge male midguts, which indicates that 20HE affects more than just the ISC mitotic rate. Long-term 20HE feeding also endowed ISCs in virgin females with proliferative characteristics similar to those seen after mating. By contrast, RNAi lines that antagonized 20HE signalling in ISCs and EBs decreased gut size in mated females and suppressed mitoses in response to detergent stress. Thus, sexually dimorphic proliferative traits of ISCs are determined in part by 20HE signalling (Ahmed, 2020).

Similar to human oestrogen and progesterone, ecdysone promotes behavioural and metabolic changes that enhance female reproduction. Dose-response assays showed that 1 mM 20HE fed to virgin females activated EcR targets and ISC mitoses to similar levels to mating. Hence, this study tested whether endogenous, mating-induced 20HE activates ISCs. Indeed, mating induced a large, transient increase in ISC division and enduring gut enlargement. This was independent of genetic sexual identity. As with exogenously fed 20HE, these effects initially required EcR only in ISCs, although EcR in EBs contributed later on. Similar to exogenous 20HE, mating also induced expression of upd2 and rho, which suggests that these are normal physiological responses (Ahmed, 2020).

To confirm the source of endogenous ecdysone, ovary-specific Gal4 drivers were used to express RNAi transgenes that target the ecdysone synthesis enzymes Dib or Spo. This suppressed mating-induced ISC divisions and midgut growth, both of which could be restored by exogenous 20HE. spo mutants also failed to resize the midgut after mating, confirming these results. To learn how the gut grows in mated females, the effects on cell size and number. Depleting EcR in ECs did not reduce EC size, but mating caused a large 20HE- and EcR-dependent increase in female ISC numbers was investigated. This expansion of the stem-cell pool could cause an increase in the total number of midgut cells. These results indicate that mating-dependent ISC division, ISC expansion and gut growth are driven by 20HE signalling from the ovaries to progenitor cells in the gut (Ahmed, 2020).

Gut growth after mating is expected to increase the absorption of nutrients by the intestine and the supply of nutrients to other organs. Because egg production is limited by nutrient availability to the ovaries, whether 20HE-dependent gut growth affected female fecundity was tested. When gut resizing was blocked by expressing EcR RNAi (EcRRNAi) in midgut ISCs, or in both ISCs and EBs, egg production was reduced by approximately 40%. This suggests that 20HE-dependent gut remodelling maximizes female reproductive fitness. However, it was also noticed that the Gal4 drivers were active in a small number of escort cells in the germarium of the ovary, which raises the possibility that these fecundity defects were due in part to a requirement for EcR in those cells (Ahmed, 2020).

A study of Drosophila juvenile hormone (JH), a sesquiterpenoid, came to conclusions similar to the current study-namely that JH promotes mating-dependent gut growth and fecundity in females. The relative roles of 20HE and JH were therefore invstigated. The JH receptors Gce and Met are essential for ISC divisions in response to not only the JH receptor agonist, methoprene, but also 20HE and infection. The mitogenic effects of methoprene were confirmed, but these were weaker than those of 20HE or mating. Furthermore, it was discovered that methoprene-stimulated divisions require 20HE, and that JH or methoprene could suppress ISC divisions driven by 20HE or other stimuli. Although these results indicate interaction between 20HE and JH, further work is required to determine their precise physiological relationship (Ahmed, 2020).

To better understand how ecdysone activates ISCs, two known EcR targets were tested: the transcription factor Broad, and the nuclear receptor Eip75B, a homologue of human PPARγ and REV-ERB17. Eip75B and broad (br) mRNA were induced in midguts by 20HE or mating, and progenitor-cell-specific depletion of either factor suppressed 20HE-induced mitoses. ISC clonal growth, however, required Eip75B but not br, highlighting that Eip75B is a more essential effector. Overexpression of Eip75B was sufficient to promote ISC division and gut epithelial turnover, whereas Eip75B loss impaired both ISC mitoses and maintenance. Progenitor-specific loss of Eip75B also blocked gut growth after mating, and compromised egg production, phenocopying the effects of EcR loss. Eip75B binds DNA to repress target genes, and also binds the nuclear receptor Hr3 to inhibit Hr3-mediated transcriptional activation. Consistent with this mechanism, overexpression of Eip75B or 20HE feeding suppressed an Hr3 activity reporter, and Hr3 overexpression suppressed ISC proliferation. Moreover, depletion of Hr3 counteracted losses in ISC proliferation caused by Eip75B depletion. Although these results indicate that Hr3 is a crucial Eip75B effector, Hr3 loss was not sufficient to activate ISC division, which indicates that Eip75B has other targets. Further tests revealed that Eip75B and Hr3 mediate 20HE-independent ISC responses to stress. Enteric infection strongly induced levels of Eip75B mRNA and suppressed Hr3 activity. Removing Eip75B or Broad from ISCs by mutation or by RNAi blocked infection-induced ISC mitoses, as did overexpression of Hr3. Eip75B was also required for ISC mitoses in response to the oxidative stress agent paraquat. Furthermore, evidence was ontained consistent with previous work that the action of Eip75B is modulated by haem (a Eip75B ligand) and nitric oxide. Functions for haem and nitric oxide in the fly gut are unknown, but potentially interesting. It is concluded that Eip75B, Broad and Hr3 integrate several inputs in addition to 20HE to control ISC proliferation (Ahmed, 2020).

As females age, they experience progressive gut dysplasia in which ISCs overproliferate and mis-differentiate, leading to high microbiota loads (dysbiosis), barrier breakdown and decreased lifespan. Age-dependent intestinal dysplasia is more pronounced in females than in males, and can be identified by increases in mitoses and mis-differentiated cells doubly positive for ISC and EC markers. Suppressing EcR, Usp or Eip75B in midgut progenitors significantly reduced both parameters of dysplasia in aged flies. Similarly, suppressing ecdysone synthesis enzymes (Dib, Spo) in the ovaries, or ubiquitously, also curtailed age-dependent gut dysplasia. This effect could be reversed by 20HE supplementation. These results indicate that age-dependent gut dysplasia is potentiated by ovary-derived ecdysone, explaining the sex bias of this condition (Ahmed, 2020).

Female Drosophila are known to be more susceptible than males to genetically induced ISC-derived tumours. This study found that ISC/EB-specific RNAi targeting Notch, a receptor required for EC differentiation, drove tumour induction in 100% of mated females but was far less tumorigenic in virgin females or males. Three results indicate that this tumour predisposition is modulated by 20HE. First, in contrast to mated females, virgins were extremely resistant to NotchRNAi-mediated tumorigenesis. Second, targeting 20HE signalling in ISCs with a dominant-negative EcR-A (EcRADN) inhibited tumour growth in mated females. Third, supplementing males or virgin females with 20HE increased tumour initiation and growth. It is surmised that the use of mating-dependent, ovary-derived 20HE to stimulate gut resizing comes at a cost: it predisposes females to gut dysplasia and tumorigenesis (Ahmed, 2020).

Gut dysplasia, tumorigenesis and egg production can all shorten lifespan, which suggests that the effects of ecdysone on the gut might adversely affect longevity. In fact, earlier reports showed that EcR mutants live longer, and proposed that reproduction can shorten lifespan by damaging the soma. The lifespan assays of this study, although subject to the same caveats as previous work, support this view: suppression of EcR in midgut progenitors extended lifespan in females but not males. In evolutionary terms, the disadvantage of a slightly shorter lifespan due to sex-specific hormonal signalling is probably insignificant relative to the reproductive fitness advantage conferred by increased egg production. This may be especially true in the wild, where gut dysplasia-dependent mortality is probably counteracted by nutrient deprivation (Ahmed, 2020).

Similarities in the reproductive biology of Drosophila and mammals suggest that these inter-organ relationships have relevance to human biology. The mitogenic effects of insect ecdysone parallel those of oestrogen and testosterone as drivers of breast, uterine and prostate growth and tumorigenesis. Yet how these steroids affect the human intestine remains poorly explored. Adaptive growth of the intestine is well documented in pregnant and lactating mammals, and might depend on oestrogen and/or progesterone. Laboratory tests with rodents and human cells, as well as some studies with human participants, have linked oestrogen, testosterone and their receptors to gastrointestinal cancers, but epidemiological studies provide conflicting evidence regarding this association. The contributions of sex steroids to intestinal physiology deserve more detailed study (Ahmed, 2020).

GENE STRUCTURE

Genomic length - 36 kb cDNA length - 5534

Bases in 5' UTR -1068

Exons - 6

Bases in 3' UTR - 1819

PROTEIN STRUCTURE

Three protein isoforms are encoded by EcR, designated ECR-A,ECR-B1 and ECR-B2. These proteins differ in their amino-terminal sequencesbut contain identical DNA binding domains and ligand binding domains. TheA and B1 isoforms are encoded by overlapping transcription units that havedifferent promotors and can be separately controlled. The N-terminal amino acids of ECR-A are coded for by three exons specific to that isoform, while both the DNA binding and ligand binding domains of ECR-A are coded for by exons shared with the other two isoforms. TheB1 and B2 isoforms are encoded by mRNAs that derive from the EcR-Bprimary transcript by alternative splicing (Talbot, 1993).

Amino Acids - 878

Structural Domains

There are two conserved domains characteristic of steroid receptor superfamilymembers. The more N-terminal domain is a DNA-binding domain and the moreC-terminal domain is a hormone-binding domain also implicated as a proteininteraction domain (Koelle, 1991). EcR is a Class II member of the nuclearreceptor superfamily, classified as such on the basis of its ability toheterodimerize with RXR (in Drosophila Ultraspiracle) and a its abilityto bind to direct repeats. EcR is most closely related to the vertebrateFarnesoid X receptor (Mangelsdorf, 1995).

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

date revised: 18 April 2024 

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