logo Metamorphosis and development of the adult fly

Formation of the adult fly
Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Vesicle-mediated steroid hormone secretion in Drosophila melanogaster
Broad specifies pupal development and mediates the 'status quo' action of juvenile hormone on the pupal-adult transformation in Drosophila
Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development
Silencing D. melanogaster lgr1 impairs transition from larval to pupal stage
The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster
Rescheduling behavioral subunits of a fixed action pattern by genetic manipulation of peptidergic signaling Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila
Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis
Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis
Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila
DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase
bantam miRNA promotes systemic growth by connecting insulin signaling and ecdysone production
Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions
Eclosion gates progression of the adult ecdysis sequence of Drosophila
Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster
CDK8-Cyclin C mediates nutritional regulation of developmental transitions through the Ecdysone receptor in Drosophila
Smads and insect hemimetabolan metamorphosis

Formation of the adult fly

Developmental origin of adult structures

With the exception of the central nervous system, most of the structures of the adult fly develop during the larval period and take their final form during eclosion, the act of emerging from the pupal case.

The familiar external structures of the adult fly develop from imaginal discs, groups of cells that invaginate from the embryonic ectoderm, and from histoblasts and imaginal rings, cells that are set aside during embryonic development and retain their ability to divide through the larval stages. The clypeus and the labrum, two anterior head structures develop from the clypeo-labral disc. The main part of the head, including the frons, the antenna, the eyes and the maxillary palps develop from the eye-antennal disc. The proboscis develops from the labial disc. The major part of the thorax, including the wing, notum, scutellum, and pleura are formed from the wing imaginal disc, the prothorax, supporting the first leg is derived from the prothoracic disc, the legs are formed from the leg discs and the haltere is formed from the haltere disc. The abdomen is formed from nests of histoblasts that are formed as part of the embryonic epidermis.

Of the internal organs, the gut, salivary glands and trachea develop from imaginal rings that are formed during embryonic development. The larval Malpighian tubules persist into adulthood. The adult musculature develops from adepithelial cells that are attached to the imaginal discs. The gonads, ovaries and testes, develop from three sources: in females only, a nest of mesodermal follicle precursor cells, and in both males and females, genital discs and pole cells (the germ line stem cells). In the female, genital discs give rise to oviducts and uterus, and the accessory structures consisting of seminal recepticle, spermathecae, and accessory glands. In the male, the genital discs give rise to seminal vesicles, ejaculatory duct and sperm pump, penis apparatus and an accessory structure, the paragonia (Hartenstein, 1993).

Events during metamorphosis

The metamorphosis of the fly begins at the end of the third instar larval stage, approximately 120 hours after the beginning of embryonic development. Studies with other insects indicates that release of Ecdysone from the ring gland is triggered by the prothoracicotropic hormone, produced by four dorsolateral neurosecretory cells of brain. Genes regulating the molting hierarchy are listed in their own site. Larval salivary gland chromosomes undergo endoreduplication and become polyploid. For information about this process, see Polytene chromosomes, endoreduplication and puffing.

Metamorphosis in Drosophila may be divided into two stages: a 12 hour prepupal period marked by pupariation (the onset of the larval-pupal transition), and a subsequent pupal period lasting 84 hours.

Pupariation is marked by a sudden release of ecdysteroid hormone secreted from the ring gland. The larval cuticle becomes the puparium or pupal case that surrounds the metamorphosing fly until it ecloses. Apolysis is the term for the retraction of the epidermis from the cuticle of the third instar larva. Once apolysis is complete, a characteristic gas bubble forms in the prepupa abdomen. At this stage the developing pupa is able to float in water.

Next the eversion of the head takes place, approximately 12 hours from the start of pupariation. The process itself is sudden, lasting about 10 minutes and orchestrated by contractions of abdominal muscles. Head eversion marks the beginning of the true pupal state. During pupariation the imaginal discs undergo eversion to form the basic shape of the adult head, thorax and abdomen. Wing, leg and haltere discs fuse to form the thorax. The eye antennal complex fuses to form a single head capsule and the head and thorax fuse with the abdomen.

During the early pupal period, final cell divisions in the wings and legs take place. Wing and leg discs inflate by the same process that drives head eversion. The next several days are marked by all the cell and tissue changes that have to take place in the development of adult structures. For example, bristles and sockets develop from precursor cells, directed by proneural genes and regulated by neurogenic genes. The layered adult cuticle develops in cycles of cuticle deposition; eye pigmentation develops and neural maturation takes place.

Early in this period a pupal to adult ecdysis takes place; for most of the pupal period the animal in the puparium is technically a pharate (cloaked or covered) adult. At the end of the pupal period eclosion (hatching) takes place, driven by an eclosion hormone (Fristrom, 1993 and Ashburner, 1989).

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo, transgenic animals were established that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of ß-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. The patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, no GAL4-LBD activation is observed in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. It is concluded that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development (Kozlova, 2002).

Spatially restricted and largely distinct patterns of GAL4-EcR and GAL4-USP activation were observed in the CNS at the onset of metamorphosis. Understanding the significance of these patterns will require more detailed studies that extend beyond the limits of this initial report. Nonetheless, there are several aspects of these activation patterns that are consistent with current understanding of the roles of EcR and USP in CNS development. (1) The cells where GAL4-EcR is most active at this stage correlate with the location of the optic proliferation zones, consistent with the known role for ecdysteroids in neuronal proliferation during metamorphosis. (2) It is also interesting to note that the pattern of GAL4-USP activation in the CNS reflects a subset of the EcR-B1 expression pattern at the onset of metamorphosis. EcR-B1 is most abundantly expressed in the mushroom body neurons and surrounding cells of the optic lobes as well as the abdominal neuromeres of the ventral nerve cord. GAL4-USP activation is strongest in a cluster of cells at the anterior end of the optic lobes that could correspond to the mushroom body neurons, and is clearly elevated in the abdominal neuromeres. (3) In addition, GAL4-USP activation in the CNS is significantly reduced in an EcR mutant background, supporting the conclusion that it is acting as a heterodimer with endogenous EcR. Interestingly, low levels of GAL4-EcR activation can also be seen in the cluster of anterior neurons in the optic lobes that show high levels of GAL4-USP activation. Unambiguous identification of these cells, however, will require more detailed studies of the patterns of GAL4-EcR and GAL4-USP activation in the CNS as well as the use of cell-type specific markers (Kozlova, 2002).

The restricted activation of GAL4-EcR cannot be attributed to the distribution of endogenous USP in the CNS, which is widely expressed in this tissue at the onset of metamorphosis. Similarly, many neurons that express EcR-B1 in the optic lobes do not show high levels of GAL4-USP activation. One possible explanation for these limited patterns of activation is that EcR might function independently of USP in certain cells of the CNS. Alternatively, any of several possible mechanisms for the reduced levels of transactivation seen in late larval imaginal discs could account for these complex cell-type specific patterns of GAL4-LBD activation in the CNS (Kozlova, 2002).

Vesicle-mediated steroid hormone secretion in Drosophila melanogaster

Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. This study presents evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland (PG) results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. It is proposed that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process (Yamanaka, 2015)

This study provides several lines of evidence demonstrating that the insect steroid hormone E is secreted from the PG not by simple diffusion, but rather through a calcium signaling-regulated vesicle fusion event. Three major points come from these findings: (1) Atet, an ABCG transporter, can facilitate E passage through membranes in an ATP-dependent manner, (2) GPCR-regulated calcium signaling in the PG promotes E release, and (3) the significance of steroid hormone release by vesicle exocytosis and its implication for other steroid hormone/cholesterol trafficking processes (Yamanaka, 2015)

Atet was originally cloned in Drosophila as an ABC transporter-encoding gene with unknown function. It was found to be highly expressed in embryonic trachea, leading to its name ABC transporter expressed in trachea or Atet. In an in situ hybridization experiment, however, this study found little expression of Atet in embryonic trachea, but instead saw specific high level expression in the PG, consistent with its expression pattern in the third instar larva. Since Atet has an atypical membrane topology and can transport E across membranes in vitro, renaming this gene Atypical topology ecdysone transporter is proposed, thereby retaining the Atet gene designation (Yamanaka, 2015)

Atet belongs to the ABCG subfamily of ABC transporters, members of which in mammals have been shown to transport cholesterol as well as other steroids, such as estrogens and their metabolites, in many biological systems. The atypical membrane topology, with the N-terminal ABC domain on the non-cytoplasmic side of the membrane, has not been reported for any ABC transporter to date. However, this topology may have a strong advantage in facilitating tight control on E release by preventing Atet from functioning on the plasma membrane, due to the lack of ATP in extracellular space. This configuration therefore prevents E transport directly through the plasma membrane and confines it to a vesicle-mediated fusion process, although it requires a separate molecular mechanism to transport ATP into the secretory vesicles. This mechanism remains unclear at this point, but it may involve a specific transporter like the recently described VNUT/SLC17A9. In this context, it is interesting to note that the human Atet orthologs ABCG1 and ABCG4 are also strongly predicted by membrane topology algorithms to position their N-terminal ABC domain on the non-cytoplasmic side. These transporters mediate cellular cholesterol efflux and have recently been shown to work not on the plasma membrane but in intracellular endosomes. Clearly, additional studies on the membrane topology of ABCG transporters are warranted (Yamanaka, 2015)

The results of the RNAi screening demonstrate that CG30054, a Gαq subunit, and Plc21C, a PLCβ class enzyme, are both required for proper PG function. These findings strongly implicate the existence of an unknown GPCR and cognate ligand as mediators of the calcium signaling event that is suggested to stimulates E release from the PG. On the other hand, it is known that the PTTH receptor is Torso, a receptor tyrosine kinase and its primary role is to promote E production by inducing E biosynthetic enzyme gene transcription. These observations suggest that, at least in Drosophila, E production and release are likely regulated separately. This machinery might help the GPCR ligand to generate large pulses of steroid in a timely fashion. The identification of the GPCR as well as its ligand is necessary to further pursue this possibility (Yamanaka, 2015)

The mechanism of steroid hormone transit through lipid membranes has not been well studied and in many physiology textbooks the issue is not even discussed. When this topic is mentioned, the explanation most often given is that they can freely diffuse through lipid membranes. Despite this prevailing assumption, there are only a few reports where such transbilayer transfer of steroids by free diffusion has been analyzed. In one theoretical study, it was shown in silico that a free energy of solvation-based mechanism can produce rapid flux of estradiol, testosterone, and progesterone through a simple membrane in concordance with measured rates. However, it is well known that steroid hormone transport across membranes can indeed be an active process in some situations: there are a number of reports on transporter involvement in either uptake or elimination of steroid hormones in eukaryotes ranging from yeast to human. These reports are suggestive enough to rationalize a potential mechanism that incorporates steroid hormones into secretory vesicles, which enables regulated secretion of steroid hormones from steroidogenic tissues (Yamanaka, 2015)

Historically, the possibility of vesicle-mediated steroid hormone release has been examined using ultrastructural and biochemical approaches in multiple biological systems, including the corpus luteum in sheep. The proposed vesicle-mediated progesterone release from the sheep corpus luteum, however, was later challenged, since the peptide oxytocin was shown to be present in dense granules by immuno-EM methods and release of oxytocin and progesterone responded differently to various secretagogues. Since that time, studies investigating the possibility of vesicle-mediated steroid release in any biological system have rarely been reported. One relevant and intriguing set of studies, however, involved ultrastructural localization of E in the PG of the waxworm Galleria mellonella using immuno-EM methods. These studies suggested that E in the PG is concentrated into what appear to be secretory granules that fuse with the plasma membrane, but once again no follow up studies have been reported in the literature (Yamanaka, 2015)

In considering the various models for steroid passage through membranes, it is important to note that steroids such as progesterone, testosterone, and estradiol are significantly more hydrophobic than E. Therefore, the free energy of solvation into a lipid bilayer of E is likely to be much more positive than for sex steroids; this may preclude the use of a simple diffusion mechanism for E. In this respect, E is more similar to bile acids, which are also highly hydrophilic and need active transporters to traverse lipid bilayers. Thus, depending on their specific physiochemical properties, different steroids might use either simple passive diffusion through the plasma membrane, active transporters or some combination of these mechanisms (Yamanaka, 2015)

In summary, this work provides strong evidence that E is released from the PG by calcium-stimulated, vesicle-mediated exocytosis. Therefore, it is suggested that the prevailing 'free diffusion' model of steroid hormone secretion needs to be reconsidered. It also follows that if E uses an active export process, then the import of many hormones, in particular 20E, is also likely controlled by transporters. Given the diversity of physiological processes regulated by steroid hormones, additional characterization of the mechanisms responsible for their import and export from various cell types and tissues will have significant impact on both basic and clinical aspects of steroid hormone physiology (Yamanaka, 2015)

Broad specifies pupal development and mediates the 'status quo' action of juvenile hormone on the pupal-adult transformation in Drosophila

An understanding of the molecular basis of the endocrine control of insect metamorphosis has been hampered by the profound differences in the responses of the Lepidoptera and the Diptera to juvenile hormone (JH). In the presence of JH, there is no change in form; in the absence of JH, ecdysone causes the switching in gene expression necessary for metamorphosis, first to the pupa, then to the adult. JH therefore prevents this switching action of ecdysone and thus maintains the 'status quo' during a molt. In the Coleoptera and in Lepidoptera such as the tobacco hornworm, Manduca sexta, where the epidermis sequentially makes several larval cuticles, the pupal cuticle and finally the adult cuticle, JH prevents each of the metamorphic transitions. By contrast, in Drosophila and the other higher flies, the pupal epidermis, except for the abdomen, is derived from imaginal discs, and exogenous JH does not prevent the larval-pupal transformation, even when given throughout larval life. Nor does JH have any effect on the subsequent external adult differentiation of the head and thorax, although JH disrupts metamorphosis of the nervous and muscular systems when given during the prepupal period. However, JH application during the final larval instar or during the prepupal period prevents the normal adult differentiation of the abdomen, whose cells arise from proliferation of the histoblasts during the prepupal period (Zhou, 2002 and references therein).

In both Manduca and Drosophila, the broad (br) gene is expressed in the epidermis during the formation of the pupa, but not during adult differentiation. Misexpression of Br-Z1 during either a larval or an adult molt of Drosophila suppresses stage-specific cuticle genes and activates pupal cuticle genes, showing that br is a major specifier of the pupal stage. Treatment with a JH mimic at the onset of the adult molt causes br re-expression and the formation of a second pupal cuticle in Manduca, but only in the abdomen of Drosophila. Expression of the Br isoforms during adult development of Drosophila suppresses bristle and hair formation when induced early or redirects cuticle production toward the pupal program when induced late. Expression of Br-Z1 at both of these times mimics the effect of JH application but, unlike JH, it causes production of a new pupal cuticle on the head and thorax as well as on the abdomen. Consequently, the 'status quo' action of JH on the pupal-adult transformation is mediated by the JH-induced re-expression of Br (Zhou, 2002).

Br has long been known to be required for the onset of metamorphosis of Drosophila because the nonpupariating (npr) alleles lack all Br proteins and remain as final instar larvae. In both Drosophila and Manduca, Br transcripts and proteins are expressed prominently during the larval-pupal transformation with different isoforms showing different temporal and tissue specificities through this period and causing either activation or suppression of specific genes. For example, in the Drosophila salivary gland, the induction of Sgs-4 and L71 and the suppression of Pig-1 during the mid and late third instar require the Z1 isoform, while the later suppression of Sgs-4 at puparium formation is due to the downregulation of another transcription factor Forkhead (Fkh) by the Z3 isoform. By contrast, the Z3 isoform activates the expression of Fbp1 in larval fat bodies during the second half of the third instar, while Z2 may play a role in repressing its premature expression. Br proteins also may play a role in the regulation of chromatin structure, since they are found in over 300 sites on the salivary gland chromosomes including sites in the interband regions and in the heterochromatin (Zhou, 2002).

Br-Z1 is the predominant isoform during the time of pupal cuticle formation in Drosophila. Whenever Br-Z1 is expressed during an ecdysone-induced molt, it can direct the epidermis into a program of pupal cuticle production. For example, the molt to the third larval instar in Drosophila begins with the rise of the ecdysteroid that peaks about 12 hours after ecdysis to the second instar. Shortly thereafter, mRNAs for larval cuticular proteins are upregulated. Expression of Br-Z1 during this time suppresses the larval cuticle gene Lcp65A-b and prematurely activates the pupal cuticle gene Edg78E. The ability of Br to be a pupal specifier is also evident during an adult molt. This molt begins about 24 hour APF with the rise of the ecdysteroid titer, and adult procuticle deposition begins about 53 hours APF during the decline of the ecdysteroid titer. Br-Z1 is most effective in activating pupal cuticle genes and suppressing an adult cuticle gene when expressed just before the normal onset of adult procuticle gene expression. This temporal restriction suggests that although Br selects which cuticle genes will be expressed, it can only do so within the confines of an ecdysone-induced program that determines the timing of cuticle gene expression at every molt. Therefore, in either a larval or an adult molt, the expression of Br-Z1 is sufficient to redirect that molt towards the pupal program (Zhou, 2002).

Adult differentiation of the epidermis can be divided into two developmental phases: cellular morphogenesis followed by cuticle deposition. Morphogenesis of the epidermis begins with the formation and outgrowth of the bristles (macrochaetes, microchaetes) between 30 and 45 hours APF, first in the head and thorax, then in the abdomen. Trichomes (hairs) are then formed by most of the general epidermal cells, beginning on the wing at 33 hours APF and on the abdomen about 48 hours APF. The general epidermis deposits three cuticular layers: cuticulin, epicuticle and procuticle. The bristle and hair shafts lack the procuticle layer. Cuticulin formation begins in patches on the wings and legs at 35-36 hours and on the abdomen at 40-45 hours APF, followed by synthesis of a continuous epicuticle once morphogenesis is complete. Adult procuticle synthesis occurs primarily between 53 and 90 hours APF. The expression of the adult cuticle gene Acp65A is restricted to flexible cuticle regions of the abdomen, the wing hinges, leg joints and the ptilinum and begins about 55-60 hours APF (Zhou, 2002).

Br disappears before the onset of adult differentiation in both Manduca and Drosophila. This disappearance is crucial for normal adult development since the misexpression of Br in Drosophila can affect both adult morphogenesis and adult cuticle production. When expressed between 30 and 40 hours APF, Br causes truncation of the bristles with early times affecting the bristles of the head and thorax and slightly later times affecting those of the abdomen. This timing corresponds to the onset of bristle outgrowth in the different regions. Suppression of bristle outgrowth occurs with misexpression of each of the Br isoforms, although the Z1 isoform has the strongest effects because the truncation is seen with expression of only two copies as well as with four copies of Br-Z1. Bristle outgrowth occurs by extension of the longitudinal actin microfilament arrays that surround the microtubular core. These actin filaments are bundled together, then crosslinked to support the elongating bristles, using sequentially the product of the forked gene and fascin. Although an occasional forked bristle is seen after misexpression of Br, the primary effect is truncation similar to that seen after exposure to inhibitors of microfilament elongation, indicating Br may be able to interfere with this process, either directly or indirectly (Zhou, 2002).

Trichome production in the abdominal epidermis is suppressed by Br-Z1 expression between 36 and 39 hours APF. Since nearly every epidermal cell normally produces a trichome, this result shows that early Br expression also suppresses morphogenesis of the general epidermis. In this case, the effective time is about 10-12 hours before abdominal trichome production. By 42 hours APF bristle and trichome morphogenesis is no longer affected by expression of Br. Between this time and 60 hours APF, the effects are primarily on the types of cuticle proteins produced. Br-Z1 is most effective in suppressing adult cuticle gene expression and causing re-induction of pupal cuticle gene expression with the resultant formation of a thin, transparent, pupal-like cuticle by the general epidermis. None of the other isoforms have such a dramatic effect on the external appearance of the cuticle, although Br-Z2 causes re-expression of the two pupal cuticle genes, and Br-Z3 causes re-expression of one pupal cuticle gene and suppression of the adult cuticle gene studied, indicating that they normally play a role in production of pupal cuticle. Cuticle is composed of many proteins, so a predominance of adult cuticle proteins could maintain the cuticular morphology despite the presence of some pupal cuticle proteins or the absence of specific adult cuticle proteins. Further study is required to resolve this issue (Zhou, 2002).

Although bristle morphogenesis is unaffected by expression of Br during the onset of cuticle formation, bristle pigmentation and sclerotization are subsequently inhibited. Whether this suppression is due to the type of epicuticle deposited or to an inhibitory action of Br on the melanization and sclerotization pathways themselves is unclear. In the case of Br-Z1, this effect is most pronounced when expression is either between 43 and 48 hours APF or later during 54-60 hours APF. Although the pupal cuticle genes used in this study all encode proteins found in the pupal exocuticle (the outer procuticle), Br-Z1 probably also directs pupal epicuticle production. If so, the earlier expression of Br-Z1 may be suppressing the deposition of the proenzymes necessary for tanning and melanization that are normally associated with adult cuticle. Such a suppression would not be unexpected, since normal pupal cuticle does not tan or melanize. These proenzymes are often laid down very early in formation of the new cuticle. Br-Z3 or Br-Z4 also suppresses bristle pigmentation but only when expressed late between 52 and 60 hours APF. This effect of later expression of any of these three isoforms is probably due to an interference with the production or deposition of the substrates for these enzymes, which normally appear in the cuticle shortly before the proenzymes are activated. However, an effect on the pigmentation process itself that occurs later cannot be ruled out (Zhou, 2002).

These different effects of Br misexpression depending on its timing indicate that Br and/or the unknown proteins whose expression Br regulates must be present to direct the pupal program. Once they disappear, the cells can revert back to the expression of the adult program. In these experiments, Br transcripts disappear by 6 hours after the heat shock, but the proteins are present until at least 9 hours. Thus, in order to obtain a second pupal cuticle that lacks bristles and trichomes, one must express Br-Z1 during both the initiation of bristle outgrowth and the onset of procuticle formation (Zhou, 2002).

An important finding of these studies is the fact that the presence of Br-Z1 at the time of cuticle formation is sufficient to redirect the program of cuticle gene expression into a pupal mode in cells that have completed their adult morphogenesis. This is most clearly seen after expression of Br at 48 or 52 hours APF. The cells of the general abdominal epidermis make the adult hairs but then deposit procuticle that includes pupal cuticle proteins. Thus, cells already committed to and expressing aspects of adult differentiation are plastic and can be caused to re-express pupal products when given the proper transcription factor. Clearly the suppression of br through the duration of adult development is essential for the normal completion of metamorphosis (Zhou, 2002).

JH has long been known to prevent metamorphosis without interfering with the molting process itself. In both Manduca and Drosophila abdomens, JH causes the formation of a second pupal cuticle only when given before the onset of the adult molt. These studies have revealed that this re-expression of the pupal program in both species is associated with the re-induction and maintenance of Br expression during the molt. This renewed Br expression appears to be sufficient to mediate the 'status quo' action of JH, since Br can both activate pupal genes and suppress adult genes. Thus, during the crucial period of adult commitment, ecdysone in the absence of JH must switch off Br so that the adult-specific program of differentiation can occur (Zhou, 2002).

In Drosophila the JH-sensitive period of the abdomen is during the prepupal period with the highest sensitivity being at the time of pupariation and loss of sensitivity after head eversion at 12 hours APF. During this time the histoblasts are proliferating rapidly. After this JH-sensitive period is over, beginning about 15 hours APF, these imaginal cells spread over the pupal abdomen and replace most of the larval cells by about 28 hours APF. Throughout this period, both types of cells express Br. JH given at pupariation has no apparent effect on the proliferation or spreading of these cells or on their replacement of the larval epidermis. Nor does it interfere with their normal Br expression during this period. Its effect is only to cause renewed and sustained expression of Br in the imaginal cells during the adult molt up through 72 hours APF (Zhou, 2002).

JH at pupariation has no apparent effect on the adult development of the Drosophila head and thoracic structures that are derived from the imaginal discs. This study shows that the refractoriness of the head and thorax to the JH treatment is due to the inability of JH to cause Br re-expression in these regions during the adult molt. Yet appropriate misexpression of Br during adult differentiation results in pupal cuticle formation in both the head and thorax, showing that Br retains its pupal-specifying function in these regions. Hence, the refractoriness to JH of the head and thorax must be due to a lesion in the pathway from the JH receptor to Br re-induction, possibly to the loss of the receptor itself (Zhou, 2002).

In all insects including Drosophila, JH is present during the larval molts, then declines during the last larval instar. In both Manduca and Drosophila epidermis and imaginal discs, Br is not expressed during the larval molt. Pupal commitment of the polymorphic epidermis of Manduca by 20E at the end of the larval feeding period is correlated with the appearance of Br, and both can be prevented by JH. By contrast, in Manduca wing imaginal discs, Br appears earlier in the final larval instar as the discs become competent to metamorphose, and JH cannot prevent this appearance but only delays it. In Drosophila and the higher flies, the pupa is derived from imaginal discs except for the abdominal cuticle that is produced by the persisting larval epidermal cells and the histoblasts. Although the effect of JH on the appearance of Br in Drosophila discs and larval epidermis has not been directly studied, dietary JH throughout larval life delays the onset of metamorphosis but does not prevent pupation, indicating that these tissues can turn on Br despite the presence of JH. Thus, the derivation of the Drosophila pupa from primarily imaginal discs probably accounts for the inability of JH to prevent the larval-pupal transformation, although the lack of effect of JH on the abdominal epidermis in its switch to pupal cuticle production remains unexplained. The mechanism whereby JH prevents the switching-on of Br by ecdysone during a larval molt and also prevents its switching-off by ecdysone at adult commitment is still unclear (Zhou, 2002).

These studies demonstrate for the first time that by the misexpression of a single transcription factor of the ecdysone cascade, the Br-Z1 isoform, one can redirect cells undergoing either larval or adult differentiation into a pupal developmental program. They also provide the first molecular basis for the 'status quo' action of JH on the pupal-adult transformation, by showing that JH causes the re-induction of Br expression and consequently re-expression of the pupal program during the molt (Zhou, 2002).

Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development

Many of the 21 members of the nuclear receptor superfamily in Drosophila are transcriptionally regulated by the steroid hormone ecdysone and play a role during the onset of metamorphosis, including the EcR/USP ecdysone receptor heterodimer. The temporal patterns of expression for all detectable nuclear receptor transcripts were examined throughout major ecdysone-regulated developmental transitions in the life cycle: embryogenesis, a larval molt, puparium formation, and the prepupal-pupal transition. An unexpected close temporal relationship was found between DHR3, E75B, and betaFTZ-F1 expression after each major ecdysone pulse examined, reflecting the known cross-regulatory interactions of these genes in prepupae and suggesting that they act together at other stages in the life cycle. In addition, E75A, E78B, and DHR4 are expressed in a reproducible manner with DHR3, E75B, and betaFTZ-F1, suggesting that they intersect with this regulatory cascade. Finally, known ecdysone-inducible primary-response transcripts are coordinately induced at times when the ecdysteroid titer is low, implying the existence of novel, as yet uncharacterized, temporal signals in Drosophila (Sullivan, 2003).

Total RNA was isolated from two independent collections of embryos staged at 2-h intervals throughout the 24 h of Drosophila embryonic development. Five Northern blots were prepared using equal amounts of RNA from each time point. These blots were sequentially hybridized, stripped, and rehybridized with radioactive probes derived from each of the 21 nuclear receptor genes encoded by the Drosophila genome. This approach allowed the generation of time courses of nuclear receptor gene expression that could be directly compared between family members. The transcripts detected are consistent with reported sizes. Transcripts from eight nuclear receptor genes were not detectable during embryonic development: E75C, E78, CG16801, DHR38, DHR83, dsf, eg, and svp (Sullivan, 2003).

Transcripts from nine nuclear receptor genes can be detected at the earliest time point (0-2 h): usp, EcR-A, FTZ-F1, DHR39, DHR78, DHR96, dERR, dHNF-4, and tll. This expression is consistent with the known maternal contribution of usp, EcR, and FTZ-F1. The observation that transcripts from DHR39, DHR78, DHR96, dERR, and dHNF-4 are undetectable by the next time point examined (2-4 h) suggests that these mRNAs are maternally loaded and rapidly degraded. EcR-B and usp transcripts are induced in early embryos, up-regulated at 6-8 h after egg laying (AEL), and maintain expression through the end of embryogenesis, with down-regulation of EcR-B in late embryos. EcR-A, in contrast, is expressed for a relatively brief temporal window, at 8-14 h AEL (Sullivan, 2003).

Six nuclear receptor genes are expressed in brief intervals during midembryonic stages. DHR39 and E75A are initially induced at 4-6 and 6-8 h AEL, respectively, and peak at 8-12 h AEL. This is followed by induction of DHR3, DHR4, and E75B at 8-12 h AEL, followed by ßFTZ-F1 expression at 12-18 h AEL. DHR39 appears to exhibit an expression pattern reciprocal to that of ßFTZ-F1, with lowest levels of mRNA at 14-16 h AEL and reinduction at 16-18 h as ßFTZ-F1 is repressed. This is followed by a second peak of E75A transcription at 18-22 h AEL (Sullivan, 2003).

A second group of nuclear receptors, DHR78, DHR96, dHNF-4, and dERR, is more broadly expressed at low levels throughout embryogenesis. DHR78 accumulates above its constant low level of expression between 8 and 14 h AEL. dERR exhibits an apparent mRNA isoform switch between 14 and 18 h AEL. dHNF-4 regulation also appears complex, with two size classes of mRNA induced at approximately 8-10 h AEL. While the 4.6-kb dHNF-4 mRNA is expressed throughout embryogenesis, the 3.3-kb mRNA is down-regulated at 14-16 h AEL. This timing is consistent with observations that dHNF-4 is expressed primarily in the embryonic midgut, fat body, and Malpighian tubules. Finally, nuclear receptors known to exert essential functions in patterning the early embryo, tll, kni, and knrl, are expressed predominantly during early stages (Sullivan, 2003).

Two genes that are not members of the nuclear receptor superfamily, BR-C and E74, were also examined in this study, as transcriptional markers for ecdysone pulses during development. Unexpectedly, both of these genes are induced late in embryogenesis, several hours after the rise in ecdysone titer at 6 h AEL. An approximately 7-kb BR-C transcript is induced at 10-12 h AEL and is present through the end of embryogenesis while E74B is induced at 14-16 h AEL and repressed as E74A is expressed from 16-20 h AEL. This BR-C expression pattern is consistent with the identification of the BR-C Z3 isoform in specific neurons of the embryonic CNS (Sullivan, 2003).

First-instar larvae were synchronized as they molted to the second instar, aged and harvested at 4-h intervals throughout second-instar larval development. Two Northern blots were prepared using equal amounts of total RNA isolated from a single collection of animals. Each blot was sequentially hybridized, stripped, and rehybridized to detect nuclear receptor transcription. The following transcripts were not detectable during the second instar: E75C, dERR, CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).

EcR-B expression is induced in mid-second-instar larvae, but does not reach maximum levels until 68-72 h AEL, just before the molt. In contrast, usp is expressed throughout the instar. A sequential pattern of nuclear receptor expression is observed that resembles the pattern seen in midembryogenesis. DHR39 and E75A are expressed in the early second instar. This is followed by induction of E75B, E78B, DHR3, and DHR4, followed by expression of ßFTZ-F1 at the end of the instar. DHR39 again shows a pattern that is approximately reciprocal with ßFTZ-F1, with highest levels during the first half of the instar. Similarly, DHR78, DHR96, and dHNF-4 exhibit broad expression patterns throughout second-instar larval development. E74A, E75A, and DHR38 are coordinately up-regulated with EcR-B at the end of the instar, between 64-72 h AEL. Finally, an approximately 9-kb BR-C transcript is detected throughout the second-larval instar (Sullivan, 2003).

Nuclear receptor gene expression was also examined throughout the third larval instar and into the early stages of metamorphosis, encompassing the ecdysone-triggered larval-to-prepupal and prepupal-to-pupal transitions. Third-instar larvae were staged relative to the molt from the second instar and harvested at 4-h intervals throughout the 48 h of the instar. Prepupae were synchronized relative to puparium formation (±15 min) and harvested at 2-h intervals up to 16 h after puparium formation (APF). Total RNA was isolated from whole animals and analyzed by Northern blot hybridization. Five blots were prepared from two independent collections of animals. These blots were sequentially hybridized, stripped, and rehybridized to detect nuclear receptor gene expression. The following transcripts were not detectable during third-instar larval or prepupal stages: CG16801, DHR83, dsf, eg, svp, tll, kn, and knrl (Sullivan, 2003).

Most nuclear receptor genes show little or no detectable expression in early and mid-third-instar larvae, a time when the ecdysone titer is low. Similar to the pattern seen in second-instar larvae, usp is expressed at relatively low levels throughout the instar and up-regulated at puparium formation, while EcR-B is induced at approximately 100 h AEL and rapidly down-regulated at puparium formation. This is followed by a sequential pattern of nuclear receptor expression similar to that seen at earlier stages. DHR39, E75A, and E78B are induced at 116-120 h AEL, in concert with the late larval ecdysone pulse, followed by maximum accumulation of E75B, DHR3, and DHR4 at 0-4 h APF. ßFTZ-F1 is expressed from 6-10 h APF, with a pattern that is approximately reciprocal to that of DHR39. EcR-A is expressed in parallel with E75B, DHR3, and DHR4 in midprepupae, similar to their coordinate expression during embryogenesis (Sullivan, 2003).

DHR78, DHR96, and dHNF-4 continue to exhibit broad expression profiles throughout third-instar larval and prepupal development. An E75 isoform not detected in embryos or second-instar larvae, E75C, is also detectable at low levels throughout most of the third instar and up-regulated in correlation with the late-larval and prepupal pulses of ecdysone. DHR38 is detectable at very low levels in early third-instar larvae, in synchrony with the early induction of E74B and BR-C. E74B is repressed, E74A is induced, and BR-C transcripts are up-regulated in late third-instar larvae, in synchrony with the late-larval ecdysone pulse. The prepupal pulse of ecdysone occurs at 10-12 h APF, marking the prepupal-to-pupal transition. EcR-A, E75A, E78B, DHR4, dERR, E75C, dHNF-4, and E74A are all induced at 10-12 h APF, in apparent response to this hormone pulse. These results are consistent with a microarray analysis of gene expression at the onset of metamorphosis where the temporal profiles of about half of these genes have been reported (Sullivan, 2003).

Most nuclear receptors can be divided into one of four classes based on this study: (1) those that are expressed exclusively during early embryogenesis (kni, knrl, tll); (2) those that are expressed throughout development (usp, DHR78, DHR96, dHNF-4); (3) those that are expressed in a reproducible temporal cascade at each stage tested (E75A, E75B, DHR3, DHR4, FTZ-F1, DHR39), and (4) those that are undetectable in these assays (CG16801, DHR83, dsf, eg, svp) (Sullivan, 2003).

Three nuclear receptor genes appear to be expressed exclusively during early embryogenesis: kni, knrl, and tll. This restricted pattern of expression fits well with the functional characterization of these genes, which have been shown to act as key determinants of embryonic body pattern. Eight genes (usp, EcR, FTZ-F1, DHR39, DHR78, DHR96, dERR, and dHNF-4) were identified that appear to have maternally deposited transcripts and thus possible embryonic functions. Indeed, maternal functions have been defined for usp, EcR, and alphaFTZ-F1 (Sullivan, 2003).

Four nuclear receptor genes are broadly expressed through all stages examined: usp, DHR78, DHR96, and dHNF-4. dHNF-4 mRNA is first detectable at 6-10 h AEL, as the ecdysone titer begins to rise. In addition, peaks of dHNF-4 expression are seen at 0, 12, and 16 h APF, in synchrony with the E74 and E75C early ecdysone-inducible genes. These observations raise the interesting possibility that this orphan nuclear receptor is regulated by ecdysone (Sullivan, 2003).

DHR38 transcripts are difficult to detect in these assays. This is consistent with studies which used RT-PCR or riboprobes for this purpose. Nonetheless, DHR38 mRNA can be detected during third-instar larval development, consistent with the widespread expression reported in earlier studies. DHR38 expression peaks at late pupal stages, consistent with its essential role in adult cuticle formation (Sullivan, 2003).

dERR and E75C display related temporal profiles of expression that do not fit with other nuclear receptor genes described in this study. Both of these genes are specifically transcribed during prepupal development, with increases in expression at 0 and 10-12 h APF. dERR, but not E75C, is also expressed during embryogenesis, with an initial induction at approximately 6 h AEL. These increases occur in synchrony with ecdysone pulses, suggesting that these orphan nuclear receptor genes are hormone inducible, although in a stage-specific manner. Further studies of dERR regulation, as well as a genetic analysis of this locus, are currently in progress (Sullivan, 2003).

Interactions between the DHR3 and E75B orphan nuclear receptors contribute to appropriate ßFTZ-F1 regulation during the onset of metamorphosis. DHR3 is both necessary and sufficient to induce ßFTZ-F1 and appears to exert this effect directly, through two response elements in the ßFTZ-F1 promoter. E75B can heterodimerize with DHR3 and is sufficient to block the ability of DHR3 to induce ßFTZ-F1. These three factors thus define a cross-regulatory network that contributes to the timing of ßFTZ-F1 expression in midprepupae. ßFTZ-F1, in turn, acts as a competence factor that directs the appropriate genetic and biological responses to the prepupal pulse of ecdysone. The patterns of DHR3, E75B, and ßFTZ-F1 expression observed at the onset of metamorphosis are consistent with these regulatory interactions as well as the expression patterns reported in earlier studies (Sullivan, 2003).

Unexpectedly, the tight linkage of DHR3, E75B, and ßFTZ-F1 expression seen at the onset of metamorphosis is recapitulated at earlier stages, after each of the major ecdysone pulses examined, in midembryogenesis and second-instar larval development. This observation suggests that the regulatory interactions between these receptors is not restricted to metamorphosis, but rather may recur in response to each ecdysone pulse during development. It is possible that this regulatory cascade contributes to cuticle deposition, which is dependent on ecdysone signaling in embryos, larvae, and prepupae. In support of this proposal, DHR3 and ßFTZ-F1 mutants exhibit defects in larval molting, suggesting that they act together to regulate this early ecdysone response (Sullivan, 2003).

Three other orphan nuclear receptor genes, E75A, DHR4, and DHR39, are expressed in concert with DHR3, E75B, and ßFTZ-F1, after the embryonic, second-instar, and third-instar ecdysone pulses. A peak of E75A expression marks the start of each genetic cascade, correlating with the rising ecdysone titer in 6- to 8-h embryos, the first half of the second instar, and in late third-instar larvae. This is followed by DHR3, E75B, and DHR4 expression which, in turn, is followed by a burst of ßFTZ-F1 expression. E78B is expressed in synchrony with DHR4 in late second and third-instar larvae, but not in embryos. These patterns of expression raise the interesting possibility that E75A, DHR4, and E78B may intersect with the cross-regulatory network defined for DHR3, E75B, and ßFTZ-F1. E75B and E78B are related to the Rev-erb vertebrate orphan nuclear receptor and are both missing their DNA binding domain. E75B and E78B null mutants are viable and fertile, suggesting that they exert redundant regulatory functions. E75A mutants die during larval stages, with no known direct regulatory targets. DHR4 mutants have not yet been described, although recent work indicates that this gene exerts essential roles in genetic and biological responses to the late larval ecdysone pulse. Further functional studies of these nuclear receptor genes should provide insight into their possible contribution to the regulatory circuit defined by DHR3, E75B, and ßFTZ-F1 (Sullivan, 2003).

Interestingly, DHR39 displays a reproducible pattern of expression that is inversely related to that of ßFTZ-F1, defining possible repressive interactions. DHR39 and ßFTZ-F1 have a similar DNA binding domain (63% identity) and bind to identical response elements, suggesting that they may exert cross-regulatory interactions. Moreover, DHR39 can repress transcription through the same response element that is activated by ßFTZ-F1. It would be interesting to determine whether the reciprocal patterns of DHR39 and ßFTZ-F1 expression during development is of functional significance (Sullivan, 2003).

The transcription of BR-C, EcR, E74, and E75 has been extensively characterized during the onset of metamorphosis, due to their rapid and direct regulation by the steroid hormone ecdysone at this stage in development. Surprisingly, however, their expression appears to be disconnected from the high-titer ecdysteroid pulses during embryonic and second-instar larval stages. As expected, EcR is induced early in embryonic development, in coincidence with the rising ecdysone titer at 4-10 h AEL, with EcR-B transcripts appearing first followed by EcR-A. BR-C mRNA, however, is not seen until 10-12 h AEL and E74B mRNA is induced even later, at 14-16 h AEL, when the ecdysteroid titer has returned to a basal level. Both EcR-B and E74B are repressed from 16-20 h AEL as E74A and E75A are induced, a switch that has been linked to the high-titer ecdysone pulse in late third-instar larvae; however, this response occurs during late embryogenesis when the ecdysteroid titer is low. A similar observation has been made for E75A expression in the Manduca dorsal abdominal epidermis, where a brief burst of E75A mRNA is detected immediately before pupal ecdysis, after the ecdysteroid titer has returned to basal levels (Sullivan, 2003).

It thus seems likely that the second instar ecdysone pulse occurs during the first half of the instar. This profile is consistent with the early induction of E75A. EcR-B and E74A, however, are not induced until the second half of the second instar, with a peak at the end of the instar. BR-C mRNA levels remain steady throughout the second instar. Finally, EcR-B, E74B, and BR-C are induced in early to mid-third-instar larvae, a time when one or more low-titer ecdysone pulses may occur. It is curious that E74B is poorly expressed relative to E74A during embryonic and second-instar larval stages, disconnecting its expression from that of EcR. This pattern is not seen in studies that focused on the onset of metamorphosis. Taken together, the temporal profiles of early gene expression (EcR, BR-C, E74, E75A) during late embryonic and late second-instar larval stages appear to be unlinked to the known ecdysteroid pulses at these stages. This could indicate that these promoters are activated in a hormone-independent manner at these stages in the life cycle. Alternatively, these ecdysone primary-response genes may be induced by a novel temporal signal that remains to be identified (Sullivan, 2003).

Several lines of evidence indicate that 20-hydroxyecdysone is not the only temporal signal in Drosophila. A major metabolite of this hormone, 3-dehydro-20-hydroxyecdysone, was shown to be as effective as 20-hydroxyecdysone in inducing target gene transcription in the hornworm, Manduca sexta. Similarly, 3-dehydro-20-hydroxyecdysone is more efficacious than 20-hydroxyecdysone in inducing Fbp-1 transcription in the Drosophila larval fat body. A high-titer pulse of alpha-ecdysone, the precursor to 20-hydroxyecdysone, can drive the extensive proliferation of neuroblasts during early pupal development in Manduca. This is the first evidence that alpha-ecdysone is responsible for a specific response in insects. It is unlikely, however, that this signal is transduced through the EcR/USP heterodimer, which shows only very low transcriptional activity in response to this ligand. Rather, recent evidence indicates that alpha-ecdysone may activate DHR38 through a novel mechanism that does not involve direct hormone binding (Sullivan, 2003).

Studies of ecdysteroid-regulated gene expression in Drosophila have also provided evidence for hormone signaling pathways that may act independently of 20-hydroxyecdysone. Several studies have identified a large-scale switch in gene expression midway through the third larval instar, an event that has been referred to as the mid-third-instar transition. It is not clear whether this response is triggered by a low-titer ecdysteroid pulse, another hormonal signal, or in a hormone-independent manner. Similarly, the let-7 and miR-125 micro-RNAs are induced at the onset of metamorphosis in Drosophila in tight temporal correlation with the E74A early mRNA, but not in apparent response to 20-hydroxyecdysone. These studies indicate that 20-hydroxyecdysone cannot act as the sole temporal regulator during the Drosophila life cycle (Sullivan, 2003).

Silencing D. melanogaster lgr1 impairs transition from larval to pupal stage

G protein-coupled receptors (GPCRs) play key roles in a wide diversity of physiological processes and signalling pathways. The leucine-rich repeats containing GPCRs (LGRs) are a subfamily that is well-conserved through[Epub ahead of print]out most metazoan phyla and have important regulatory roles in vertebrates. This study reports on the critical role of Drosophila melanogaster LGR1, the fruit fly homologue of the vertebrate glycoprotein hormone receptors, in development as a factor involved in the regulation of pupariation. Transcript profiling revealed that lgr1 transcripts are most abundant in third instar larvae and adult flies. The tissues displaying the highest transcript levels were the hindgut, the rectum and the salivary glands. Knockdown using RNA interference (RNAi) demonstrated that white pupa formation was severely suppressed in D. melanogaster lgr1 RNAi larvae. Associated with this developmental defect was a reduced ecdysteroid titer, which is in line with significantly reduced transcript levels detected for the Halloween genes shadow (sad) and spookier (spok) in the third instar lgr1 RNAi larvae compared to the control condition (Broeck, 2014).

Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). This study shows that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulated ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. The study proposes that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant (Ohhara, 2015).

In many animal species, the developmental transition is a well-known biological process in which the organism alters its body morphology and physiology to proceed from the juvenile growth stage to the adult reproductive stage. For example, in mammals, puberty causes a drastic change from adolescent to adulthood, whereas in insects, metamorphosis initiates alteration of body structures to produce sexually mature adults, a process accompanied by changes in habitat and behavior. These developmental transitions are primarily regulated by steroid hormones, production of which is regulated coordinately by developmental timing and nutritional conditions. How these processes are precisely regulated in response to developmental and environ mental cues is a longstanding question in biology (Ohhara, 2015).

In holometabolous insects, the steroid hormone ecdysone plays a pivotal role in metamorphosis. In Drosophila, metamorphic development from the third-instar larva into the adult, through the prepupa and pupa, initiates 90-96 h after hatching (hAH) at 25°C under a standard culture condition. At the onset of the larval-prepupal transition, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the peripheral organs. The activities of 20E terminate larval development and growth and initiates metamorphosis. Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during development. The best-known stimulator of ecdysone biosynthesis is prothoracico-tropic hormone (PTTH), which is produced by neurons in the CNS. PTTH activates the receptor tyrosine kinase Torso in the PG to stimulate expression of ecdysone biosynthetic genes through the Ras85D/Raf/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Insulin-like peptides (Ilps), members of another class of neuron-derived factors, activate PI3K in the PG, resulting in production of ecdysone biosynthetic proteins. The Activin/transforming growth factor-β (TGF-β) signaling pathway is also required in the PG for the expression of PTTH and Ilps receptors, although to date it remains unclear which organ produces the ligand that acts on the PG (Ohhara, 2015).

In addition to these neuropeptides, the larval-prepupal transition is modulated by environmental cues such as nutritional conditions that influence larval body size. For example, at 56 hAH, early third-instar larvae attain the minimal viable weight (MVW), at which sufficient nutrition is stored in larvae to ensure their survival through metamorphosis. After attaining MVW, larvae pass another checkpoint, critical weight (CW), defined as the minimum larval size at which starvation no longer delays the larval-prepupal transition. In Drosophila, both checkpoints occur almost simultaneously, making it difficult to distinguish them. However, CW is regarded as a body size checkpoint that initiates metamorphosis and is therefore believed to ultimately modulate ecdysone production in the PG. However, its downstream effectors and signaling pathway remain elusive (Ohhara, 2015).

Based on data obtained in Manduca and Bombyx, a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. This study shows that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval-prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. It is proposed that this autocrine system acts downstream of the CW checkpoint to allow the larval-prepupal transition. It is speculated that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions (Ohhara, 2015).

Previously studies have shown that the GPCR Octβ3R is expressed in multiple larval tissues, including the PG. To determine whether Octβ3R is involved in ecdysone biosynthesis and metamorphosis, RNAi was used to knock down Octβ3R function specifically in the PG, using the Gal4-upstream activation sequence (UAS) system. Two different UAS-Octβ3RRNAi constructs targeting distinct regions of the Octβ3R mRNA (Octβ3RRNAi-1 and Octβ3RRNAi-2) were used to knock down Octβ3R in the PG with the help of a phantom (phm)-22-Gal4 driver. Strikingly, larvae expressing Octβ3RRNAi in the PG never developed into adult flies, and 96% of phm>Octβ3RRNAi-1 animals and 34% of phm>Octβ3RRNAi-2 animals arrested at the larval stage. When UAS-dicer2 was introduced into phm>Octβ3RRNAi-2 larvae (phm>Octβ3RRNAi-2+dicer2) to increase RNAi activity, all of these animals arrested at the larval stage. Using in situ hybridization, a significant reduction was confirmed in the Octβ3R mRNA levels in the PG of knockdown animals relative to control larvae. These data suggest that Octβ3R expression in the PG is essential for executing the larval-prepupal transition in metamorphosis (Ohhara, 2015).

Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3RRNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

Octβ3R is thought to be activated by octopamine and tyramine binding. Octopamine is synthesized from tyramine by tyramine β-hydroxylase (Tbh), and tyramine is synthesized from tyrosine by tyrosine decarboxylase (Tdc). In Drosophila, two Tdc genes (Tdc1 and Tdc2) and one Tbh gene have been identified, and all of them are expressed in the larval CNS. Tdc1, Tdc2, and Tbh are also expressed in the PG. Furthermore, octopamine and tyramine were detected in the PG by immunostaining. Thus, octopamine and/or tyramine synthesized in the PG may activate Octβ3R in an autocrine manner to induce ecdysone production (Ohhara, 2015).

To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2RNAi-1 and Tdc2RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2RNAi-1+dicer2 and phm > Tdc2RNAi-2+dicer2). All phm > Tdc2RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2RNAi-1+ dicer2 and phm > Tdc2RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

To determine which Octβ3R ligand is responsible for this autocrine signaling, Tbh was knocked down in the PG to prevent conversion of tyramine into octopamine. To knock down Tbh, two constructs targeting distinct regions of the Tbh transcript (TbhRNAi-1 and TbhRNAi-2) were expressed along with dicer2 under the control of phm-22-Gal4 (phm > TbhRNAi-1+ dicer2 and phm > TbhRNAi-2+dicer2). Although the Tbh knockdown caused a reduction in octopamine production in the PG, these larvae did not exhibit any obvious defects in the larval-prepupal transition or subsequent metamorphosi. These data suggest that tyramine, rather than octopamine, is the Octβ3R ligand that activates ecdysone production in the PG (Ohhara, 2015).

Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3RRNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3RRNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3RRNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

The observations described above demonstrate that phm>Octβ3RRNAi affects Ilps and PTTH signaling in the PG as early as 60 hAH, raising the question of when Octβ3R function is required in the PG for execution of the larval-prepupal transition. To address this issue, the Gal80ts and Gal4/UAS system, which restricts expression of Octβ3R dsRNA in the PG at 18oC, but allows its expression at 28oC, was used. The results of temperature upshift and downshift experiments revealed that the larval-prepupal transition was impaired only when Octβ3R dsRNA was expressed in the PG at around 60 hAH. Notably, 60 hAH is the critical period during which larvae attain CW under nutrient-rich conditions. As noted above, when larvae are starved before attainment of CW, they are unable to transit into the prepupal stage. By contrast, starved larvae can successfully transit to prepupal/pupal stage without developmental delay once they have attained CW by growing beyond the critical period (~56 hAH) under nutrient-rich conditions in standard Drosophila medium. Thus, it is hypothesized that Octβ3R signaling acts downstream of the body-size checkpoint, or attainment of CW, to allow the larval-prepupal transition (Ohhara, 2015).

Several lines of evidence support this hypothesis. First, Octβ3R function is required for activation of Ilps and PTTH signaling detected in the PG at 60 hAH. By contrast, at 48 hAH, before the attainment of CW, neither signaling pathway is active in the PG. Second, Ilps and PTTH signaling was not activated in the PG when the larvae were starved from 48 hAH onward (early starvation), whereas these signaling pathways were active when the larvae were starved after 60 hAH (late starvation). Finally, a ligand for Octβ3R, tyramine, was detectable in the PG at 60 hAH, but decreases after this stage under a nutrient-rich condition. This decrease in tyramine was abrogated by early starvation but not by late starvation. Assuming that this decrease in tyramine in the PG is due to its secretion from PG cells, it is reasonable to propose that attainment of CW causes tyramine secretion from the PG at around 60 hAH, which in turn activates Octβ3R to regulate the Ilps and PTTH pathways, leading to the larval-prepupal transition (Ohhara, 2015).

This study demonstrates that monoaminergic regulation plays a pivotal role in ecdysone biosynthesis to induce metamorphosis and that Octβ3R acts as an upstream regulator essential for the Ilps and PTTH signaling. In addition, the data indicate that Octβ3R ligands are produced in the PG to stimulate ecdysone biosynthesis in an autocrine manner. Autocrine signaling has been proposed to mediate the community effect, in which identical neighboring cells are coordinated in their stimulation and maintenance of cell type-specific gene expression and their differentiation, as observed in muscle development of amphibian embryos. Thus, it is proposed that monoaminergic autocrine signaling among PG cells acts to increase their responsiveness to Ilps and PTTH, thereby allowing coordinated ex- pression of ecdysone biosynthetic genes within a time window following exposure to neuropeptides (Ohhara, 2015).

These findings raise the larger question of whether monoamine acts as part of an evolutionarily conserved mechanism of steroid hormone production. In vertebrates, there is limited evidence of monoaminergic regulation of steroid hormone biosynthesis. For example, in cultured adrenal glands, catecholamine stimulates the biosynthesis of the steroid hormone cortisol in a paracrine manner to elicit a stress reaction. Another example is the Leydig cells of the mammalian testes, in which the steroid hormone testosterone is produced mainly in response to pituitary gonadotropin. However, catecholamine signaling through β-adrenergic receptors, the orthologs of Octβ3R, also promotes the production of testosterone from cultured fetal Leydig cells, which may be the site of catecholamine synthesis in the fetal and mature human testes. Thus, monoamines may play a conserved role in modulating and/or stimulating steroid hormone production during physiological and developmental transitions (Ohhara, 2015).

The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster

The timely onset of metamorphosis in holometabolous insects depends on their reaching the appropriate size known as critical weight. Once critical weight is reached, juvenile hormone (JH) titers decline, resulting in the release of prothoracicotropic hormone (PTTH) at the next photoperiod gate and thereby inducing metamorphosis. How individuals determine when they have reached critical weight is unknown. Evidence is presented that in Drosophila, a component of the ring gland, the prothoracic gland (PG), assesses growth to determine when critical weight has been achieved. The GAL4/UAS system was used to suppress or enhance growth by overexpressing PTEN or Dp110 (Pi3K92E), respectively, in various components of the ring gland. Suppression of the growth of the PG and CA, but not of the CA alone, produced larger-than-normal larvae and adults. Suppression of only PG growth resulted in nonviable larvae, but larvae with enlarged PGs produced significantly smaller larvae and adults. Rearing larvae with enlarged PGs under constant light enhanced these effects, suggesting a role for photoperiod-gated PTTH secretion. These larvae are smaller, in part as a result of their repressed growth rates, a phenotype that could be rescued through nutritional supplementation (yeast paste). Most importantly, larvae with enlarged PGs overestimated size so that they initiated metamorphosis before surpassing the minimal viable weight necessary to survive pupation. It is concluded that the PG acts as a size-assessing tissue by using insulin-dependent PG cell growth to determine when critical weight has been reached (Mirth, 2005; full text of article).

These manipulations of insulin-dependent PG growth showed that this growth is inversely related to larval growth. Suppressing the growth of the PG (P0206>PTEN - ectopically driven PTEN) produced larvae that spent more time in each instar and were larger than normal. These effects are presumably due to a combination of reduced ecdysteroid biosynthesis, which is known to delay development, and increased growth rate. Conversely, larvae with enlarged PGs (phm>Dp110; phm is a phantom GAL4 line which was used to drive expression of Dp110) showed accelerated development in the L3. Their growth rate was dependent on nutritional conditions. Whereas phm>Dp110 larvae reared on suboptimal food grew slowly, well-fed phm>Dp110 larvae grew at the same rate as controls. Together, these data indicate that the growth of the PG negatively regulates the growth rate of the whole animal and that this regulation is modulated by nutrition (Mirth, 2005).

In addition, decreasing PG size in P0206>PTEN larvae resulted in premature metamorphosis and the formation of L2 puparia. Similar L2 puparia have been described in larvae with mutations that affect the regulation of ecdysteroid biosynthesis or signaling and in larvae where the Broad isoform Z3 was overexpressed in the ring gland, resulting in its apoptosis. L2 puparia are seen in situations where ecdysone synthesis is compromised because larvae cross the threshold weight for metamorphosis prior to the production of sufficient ecdysone to initiate a larval molt, redirecting their development to the metamorphic pathway (Mirth, 2005).

Reducing PG size resulted in reduced ecdysteroid biosynthesis; P0206>PTEN larvae showed reduced ecdysteroid titers at 44 hr AEL3, and phm>PTEN larvae only molted to L2 when fed 20E. Under conditions of low ecdysteroid synthesis, fast-growing larvae could surpass the threshold for metamorphosis before the ecdysteroid titer was sufficient to induce a molt, resulting in L2 prepupae. Slower-growing larvae would be unable to reach this threshold weight before the rise in ecdysteroid titer induced the molt to L3. Indeed, undernourished, and presumably slow-growing, P0206>PTEN L2 larvae all molted to L3, whereas only 33% of the well-fed P0206>PTEN larvae molted to L3 (Mirth, 2005).

Enlarging the PG of larvae reared under constant light caused larvae to initiate metamorphosis earlier and at smaller sizes. Nevertheless, even though larvae starved early after the L3 molt were able to pupariate, they were unable to survive to pupation unless they had fed for at least 11.5 hr. This suggests that phm>Dp110 larvae starved prior to 11.5 hr AL3E initiated metamorphosis before surpassing the minimal viableweight. Furthermore, although in control larvae, critical weight and minimal viable weight are apparently attained at the same time, they are uncoupled in phm>Dp110 larvae. Therefore, the assessment of critical weight is dependent on PG growth, whereas the minimal viable weight is not (Mirth, 2005).

In Drosophila, the PGs are responsible for a size-assessment event, early in the L3, that induces the onset of metamorphosis once critical weight is surpassed. Enhancing PG growth resulted in an overestimation of body size, thereby causing the larva to initiate metamorphosis early, at a subnormal size. Under LL, the effects of enlarging the PG were enhanced, producing individuals that pupariated even earlier at even smaller sizes, suggesting that when PTTH release was unconstrained by circadian gating, the PTTH delay period was reduced. These data provide the first indication in Drosophila that the post-critical-weight PTTH release may be under photoperiod control, as it is in Manduca (Mirth, 2005).

There has been some discussion in the literature as to whether critical weight as described in Drosophila is the same as critical weight as defined in Manduca. This discussion has arisen because the definition for Manduca states that critical weight is the minimal size at which starvation can no longer delay the onset of metamorphosis, but when Drosophila larvae are starved before critical weight is reached, they die. The current data suggest that this is due to a tight relationship between minimal viable weight and critical weight in Drosophila. Effects more similar to those observed in Manduca can be obtained when pre-critical-weight Drosophila larvae are starved for an interval and then re-fed. Under these conditions, they delay metamorphosis for a period greater than the period of starvation. Much of the confusion surrounding critical weight in Drosophila has arisen because in wild-type larvae, minimal viable weight and critical weight are achieved at the same time (Mirth, 2005).

After critical weight has been surpassed, the metamorphic pathway appears to be partially suppressed by continued feeding in Drosophila. Hence, the nutrition pathway appears to promote growth and suppress metamorphosis, whereas insulin-dependent PG growth suppresses larval growth and promotes differentiation (Mirth, 2005).

The effects of increased growth in the PG are not simply due to increasing cell size, but rather are specific to the nutrition-dependent InR signaling pathway. Studies have indicated that when either dMYC or cyclinD/cdk4 are used to enlarge the PG cells, there is no reduction in overall body size. Overexpression of dMYC, of cyclinD/cdk4, and of Dp110 all enhance cell growth, but they do so in fundamentally different manners by using separate cascades. Whether the size-assessment mechanism operates via increased intracellular PIP3 levels in the PG cells or the accumulation of some other downstream component of the InR cascade in these cells is unknown (Mirth, 2005).

Although no difference in was detected ecdysteroid titers in larvae with enlarged PGs, there is evidence that increased InR signaling in the PG cells can produce mild increases in ecdysteroidogenesis and ecdysone signaling, increases that are below the level of detection of ecdysteroid-titer assays. Larvae with enlarged PGs showed both a mild increase in the transcription of phantom during feeding stages and an increase in the transcription of the early ecdysone response gene E74B. These subtle differences in ecdysteroid titers may be important for determining growth rates and for size assessment. A gradual rise in ecdysteroid titers is coincident with the time that critical weight is reached in Drosophila. Also, subtle shifts in 20E concentrations are important for growth. Basal concentrations of 20E in combination with bombyxin enhance the growth of wing imaginal tissues in vitro; slightly higher concentrations of 20E suppress growth (Mirth, 2005).

Mutations that cause imaginal disc and larval overgrowth often cause delayed pupariation and, in some cases, show low L3 ecdysteroid titers. In the case of the mutant lethal (2) giant larvae, the ring glands are smaller than normal and have the ultrastructural appearance of glands that have low rates of ecdysteroid biosynthesis. Delayed pupariation in these larvae can be rescued by implanting wild-type ring glands. Lastly, hypomorphic mutations in DHR4, a repressor of ecdysone-induced early genes, cause reductions in critical weight and early-pupariation phenotypes similar to those described in this study. Thus, the size-assessment mechanism is likely to involve surpassing a threshold ecdysteroid titer above which the activation of the ecdysone cascade occurs (Mirth, 2005).

These data allow construction of the following model for size assessment in Drosophila. As PG cells grow in response to increased InR signaling, they increase their basal level of ecdysteroid biosynthesis. Critical weight is then reached when systemic ecdysteroid concentrations surpass a threshold that sets into motion the endocrine events that will end the growth phase of larval development and allow the larva to begin metamorphosis (Mirth, 2005).

Studies in the mid-1970s defined a size-assessment event during the final instar of the moth Manduca sexta; termed critical weight, it is the minimal size required for the timely initiation of metamorphosis. How insect larvae determine when they have reached critical weight has long been a mystery. It is hypothesized that a size-assessing tissue determines when critical weight had been reached. Suppressing growth in this size-assessing tissue would cause an underestimation of body size, resulting in metamorphosis at larger than normal sizes, whereas enlarging this tissue would result in subnormal sizes. Studies in Drosophila have shown that manipulation of the growth of the PG via the InR pathway produced these types of effects. Furthermore, larvae with enlarged PGs metamorphosed at even smaller sizes when reared under LL, suggesting a role for PTTH circadian gating in this response. Smaller size arose both as a result of a reduction in growth rate, an effect that could be rescued via nutritional supplementation, and the early onset of metamorphosis. Most importantly, larvae with enlarged PGs had a remarkably reduced critical weight, suggesting that they are severely overestimating their own body size. These results offer a very new perspective on the problem of size control in insects, uniting the recent data exploring the role of nutrition and the insulin-receptor pathway on growth with the classical physiological experiments that defined critical weight (Mirth, 2005).

Rescheduling behavioral subunits of a fixed action pattern by genetic manipulation of peptidergic signaling

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. This study presents evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. These findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons (Kim, 2015).

Innate behaviors are stereotypic patterns of movement inherited from birth that require no prior experience for proper execution. Among such behaviors are fixed action patterns that, once initiated, run to completion independent of sensory inputs. Examples include courtship rituals, aggression displays, and ecdysis. Ecdysis represents a 'chemically-coded' behavioral sequence triggered by peptidergic ecdysis triggering hormones (ETH), which orchestrate a downstream peptidergic cascade leading to sequential activation of central pattern generators underlying patterned motor activity. The term Fixed Action Pattern (FAP) has fallen into disuse, since innate behaviors generally exhibit considerable plasticity. However the invariant nature of the ecdysis behavioral sequence makes it a clear example a classic FAP. In depth analysis of ecdysis behavior may provide a more thorough understanding of how hormones assemble and regulate behavioral circuitry in the brain, in particular circuits that operate sequentially (Kim, 2015).

ETHs are released by peripheral peptidergic Inka cells in response to declining levels of the steroid hormone 20-hydroxyecdysone. Presence of Inka cells in more than 40 species of arthropods, along with the sequence similarity of ETH peptides in diverse insect groups, suggests that ETH signaling is highly conserved in insects. Identification of the Ecdysis Triggering Hormone receptor (ETHR) gene in Drosophila melanogaster (Park, 2002) enabled elucidation of a complex downstream signaling cascade triggered by ETH. The ETHR gene encodes two functionally distinct subtypes of G protein coupled receptors (ETHR-A and -B) through alternative splicing. The presence of two ETH receptor subtypes has been observed in all insect species thus far examined (Roller, 2010). The two receptor subtypes show differences in ligand sensitivity and specificity and are expressed in separate populations of central neurons, suggesting that they have distinctive roles in ETH signaling (Kim, 2015).

A diversity of ETHR neurons in the moth Manduca sexta and fruitfly Drosophila melanogaster has been identified (Kim, 2006a; Kim, 2006b). One of the most striking properties of ETHR-A neurons is that they are virtually all peptidergic and conserved across insect orders. Groups of ETHR-A 'peptidergic ensembles' express a range of different neuropeptides, including kinins, diuretic hormone (DH), eclosion hormone (EH), FMRFamide, crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIPs), bursicon (burs and pburs), neuropeptide F (NPF), and short neuropeptide F (sNPF). It is hypothesized that released ETH acts directly on the CNS to activate these peptidergic ensembles for control of specific central pattern generator circuits that elicit stereotyped ecdysis behaviors. However evidence for direct actions of ETH on these target ensembles is yet to be reported (Kim, 2015).

Likely functions of certain ETHR-A peptidergic ensembles in Manduca have been inferred from pharmacological manipulations (Kim, 2006b). For example, serially homologous L3/4 neurons of abdominal ganglia in Manduca express a cocktail of kinins and diuretic hormones; exposure of the isolated CNS to these peptides elicits a fictive pre-ecdysis I-like motor pattern. Similarly, the IN704 peptidergic ensemble that co-expresses CCAP and MIPs is implicated in initiation of ecdysis behavior, since co-application of these two peptides elicits fictive ecdysis behavior. Homologous peptidergic ensembles in Drosophila exhibit characteristic patterns and time courses of calcium mobilization indicative of electrical activity coincident with successive steps in the ecdysis FAP (Kim, 2006a). Of particular interest are observations that bursicon, a hormone co-expressed in a subset of CCAP neurons, is required for ecdysis behavior (Kim, 2015).

This study tested hypotheses that two central peptidergic ensembles—kinin neurons and a subset of CCAP neurons (CAMB) that co-express CCAP, Allatostatin CC, Myoinhibitory peptide, and Bursicon—are direct targets of ETH and schedule pre-ecdysis and ecdysis behavior components of the ecdysis FAP, respectively in the fruit fly Drosophila. It was shown further that manipulation of ETHR expression levels and signal transduction specifically in these ensembles influences scheduling of the FAP. Finally, possible mechanisms are described underlying timing of the switch from pre-ecdysis to ecdysis behavior and a model is proposed to explain mechanistically how these behaviors are sequentially activated (Kim, 2015).

The aims of this study were to implicate identified ETHR neuronal ensembles with specific steps in the ecdysis FAP. Through genetic manipulation of all known ETHR-A central neuron ensembles and subsets thereof, kinin and CAMB neurons were implicated in scheduling of pre-ecdysis and ecdysis, respectively. The current findings confirm that these ensembles are targeted directly by ETH. A key observation is timing of calcium mobilization in kinin and CAMB ensembles following ETH application: kinin neurons mobilize calcium within minutes, while activity in CAMB neurons is delayed. CAMB neurons mobilize calcium only after kinin neuron activity ceases, some 10 minutes later. These temporal patterns of cellular activity correspond well with those of pre-ecdysis and ecdysis behaviors observed in vivo (Kim, 2015).

The findings demonstrate that the kinin peptide ensemble is necessary for proper scheduling of pre-ecdysis behavior, if not itself sufficient to elicit it. Kinin cell ablation abolishes pre-ecdysis behavior in a significant percentage (25%) of animals. The remaining 75% of individuals showed highly variable pre-ecdysis duration, ranging from 3-22 minutes, whereas duration of this behavior in control animals is tightly regulated at 9.1 ± 0.9 min. Furthermore, a 30% reduction in Leukokinin receptor expression, caused by a piggyBac-element insertion into the promoter region of the gene, also disrupts fidelity of pre-ecdysis regulation; this phenotype is rescued by precise excision of the piggyBac insertion. Finally, RNA silencing of the kinin receptor in peripheral neurons using the Peb-Gal4 driver leads to reduced intensity of the behavior and greatly increases variability of pre-ecdysis duration. This is the first report demonstrating that kinin signaling affects scheduling of the ecdysis behavioral sequence via actions on peripheral neurons. Pan-neuronal silencing of kinin receptors also disrupted pre-ecdysis scheduling, but to a lesser extent (Kim, 2015).

Manipulation of ETHR expression levels in kinin neurons also alters scheduling of pre-ecdysis behavior significantly, confirming that these neurons are targeted directly by ETH and that they play an important role in pre-ecdysis regulation. It was reasoned that knockdown of receptor expression in these neurons would lead to a lower density of ETHR in the plasma membrane, thereby reducing sensitivity to ETH and delaying onset of pre-ecdysis. These experimental results demonstrated a reduction of pre-ecdysis duration. This reduction is attributed to a delay in pre-ecdysis onset, brought about by the need for higher ETH levels for neuronal activation. Since timing of the switch to ecdysis (controlled by CAMB neurons; see below) is unaffected, pre-ecdysis duration is shortened. On the other hand, overexpression of ETHR in kinin neurons led to prolongation of pre-ecdysis duration. Following the same reasoning, this would result from premature kinin neuron activation attributable to higher sensitivity to rising ETH levels and overall longer pre-ecdysis (Kim, 2015).

Kinins were identified originally using bioassays for myotropic and diuretic functions and they have well-known actions on muscle and transport activity in epithelia. More recent studies demonstrated diverse functional roles for kinin signaling, including feeding, olfaction, and locomotory behavior. Previous works demonstrated ETHR-A expression in kinin neurons of both Drosophila and Manduca, implicating them as direct targets of ETH. Imaging studies have shown that abdominal kinin neurons in fly larvae exhibit periodic calcium oscillations under normal conditions and are involved in turning behavior. These kinin neurons project to a terminal plexus in close association to kinin receptors, suggesting it functions as a site of peptide release. Interestingly, this same ensemble of kinin neurons in the pre-pupal preparation used here showed no such periodic activity, but instead exhibited synchronized calcium oscillations activity following exposure to ETH. This difference could be unique to the pharate stage (i.e., preceding ecdysis) animal, during which insects generally are unresponsive to external stimuli. In imaging studies, ETH-induced calcium dynamics were observed to be initiated from the terminal plexus region and subsequently move anteriorly to the cell bodies. These observations suggest that this plexus serves critical functions in both sending and receiving signals. Evidence presented in this study for regulation of pre-ecdysis behavior by kinin neurons demonstrates a new function for this peptide in Drosophila, which is reinforced by previous observations in Manduca that application of kinin causes a fictive pre-ecdysis motor pattern in the isolated CNS (Kim, 2015).

How do kinin neurons function in the promotion of pre-ecdysis behavior? While manipulation of kinin signaling clearly affects behavioral intensity and duration, this study was unsuccessful in initiating pre-ecdysis through temperature-dependent activation of kinin neurons expressing either TRPM8 and TRPA1. It is concluded that, while kinin functions as a modulatory influence necessary for proper scheduling of pre-ecdysis behavior, other as yet unidentified signals are necessary for behavioral initiation (Kim, 2015).

This study has demonstrated that CAMB neurons are both necessary and sufficient for the switch from pre-ecdysis to ecdysis behavior. This conclusion rests on results from a combination of experiments. First, CAMB cell ablation abolishes the switch to ecdysis, suggesting these neurons are necessary for the switch. Failure of ecdysis initiation is attributable to bursicon deficiency, since a previous report showed clearly that expression of the bursicon gene is required for initiation of pupal ecdysis. Calcium mobilization in CAMB neurons is delayed for ~10 min after onset of activity in kinin neurons, which fits well with the ~10 min delay before appearance of ecdysis behavior following onset of pre-ecdysis behavior observed in vivo. Altered levels of ETHR expression in CAMB neurons clearly affects timing of the switch to ecdysis behavior: receptor knockdown delays the switch, whereas overexpression accelerates it. In vitro experiments confirm that altered ETHR expression levels affect timing of calcium mobilization in CAMB neurons in register with changes in behavioral timing (Kim, 2015).

Finally, this study showed that activation of CAMB neurons through temperature-sensitive TRPM8 expression initiates ecdysis behavior in vivo. Thus, CAMB neurons are both necessary and sufficient for the switch to ecdysis behavior. However, activity in CAMB neurons alone does not result in robust ecdysis behavior. Expression of ecdysis behavior with parameters corresponding to that observed in wild-type flies requires activation of the entire CCAP ensemble (Kim, 2015).

It is interesting that, in all TRPM8 activation experiments, removal of the temperature stimulus led to re-capitulation of the entire ecdysis FAP. This might be explained by positive feedback influences on the Inka cell to release ETH, possibly via EH neurons. Alternatively, the ecdysis motor circuit may exert negative feedback on the pre-ecdysis circuit, which when removed, causes a post-inhibitory rebound leading to activation of the pre-ecdysis circuit and the entire FAP. Attempts to demonstrate such negative feedback were inconclusive in this study (Kim, 2015).

CAMB neurons express a combination of CCAP, Ast-CC, MIP, and bursicon. In Manduca, application of a CCAP/MIP cocktail is sufficient to elicit fictive ecdysis behavior. It would be parsimonious to extrapolate this result to Drosophila, since both of these peptides are found in CAMB neurons. Nevertheless, in Drosophila it is clear that bursicon is a key signaling molecule necessary for ecdysis initiation. It remains to be demonstrated precisely how absence of the bursicon gene blocks the switch to ecdysis. It will be interesting to elucidate possible functional roles of co-expressed peptides in CAMB neurons CCAP, Ast-CC, MIP in activation of the motor circuitry encoding the ecdysis motor pattern (Kim, 2015).

How is timing of the switch to ecdysis determined? Since both kinin and CAMB ensembles express ETHR, one would expect ETH to activate both ensembles simultaneously. Several previous studies provide evidence for the role of descending inhibition from cephalic and thoracic ganglia in setting the delay in the switch to ecdysis behavior. This study shows that expression of pertussis toxin in CAMB neurons accelerates the switch to ecdysis, consistent with disinhibition of Gαi/o input(s). It is hypothesized that a balance of excitatory and inhibitory inputs to the CAMB neurons contributes to the delay in their activity, excitatory input coming from ETH via Gαq signaling and Gαi/o from an as yet unidentified transmitter descending from cephalic and/or thoracic ganglia. The finding that RNAi-knockdown of MIP neurons lying outside the CCAP ensemble accelerates the switch to ecdysis behavior suggests one such possible inhibitory input (Kim, 2015).

It is possible, if not likely that ETH drives both inhibitory and excitatory inputs to CAMB neurons, with ETHR-B-expressing inhibitory inputs preceding excitatory input. Such a scenario follows from the fact that sensitivity of Drosophila ETHR-B to ETH was shown to be ~450-fold higher than that of ETHR-A. Therefore, as ETH levels rise in the hemolymph, ETHR-B-expressing inhibitory neurons would be activated well before ETHR-A neurons. ETH would effectively inhibit CAMB neurons indirectly prior to direct excitation via ETHR-A activation (Kim, 2015).

Such a scenario pre-supposes that the EC50 values governing activation of ETH receptors determined previously from heterologous expression in mammalian CHO cells are valid in Drosophila neurons. Data presented in this study suggests this is so. The EC50 value for ETH1 against ETHR-A was found to be ~414 nM, while the EC50 for ETH2 was determined to be ~4.3 μM. This study applied a combination of ETH1 and ETH2, each at a concentration of 300 nM, to the isolated CNS and obtained a pattern of calcium dynamics in kinin neurons lasting for ~10 min, which matches the duration of pre-ecdysis behavior under natural conditions. Furthermore, the switch to ecdysis behavior occurs ~10 min after initiation of calcium mobilization in kinin neurons, which corresponds to timing of the switch to ecdysis behavior in vivo. Doubling concentrations of the ETH1/ETH2 cocktail reduced the duration of calcium dynamics in kinin neurons to 5.5 min and accelerated the switch to ecdysis behavior. These results make it likely that the relative sensitivities of ETHR-B and ETHR-A are as previously established and consequently activity in ETHR-B neurons would precede that of ETHR-A neurons (Kim, 2015).

This study has shown that altered levels of ETHR expression have significant consequences for timing of pre-ecdysis duration and timing of the ecdysis switch. These findings raise the possibility that scheduling of sequential steps in the ecdysis FAP may be a consequence of different sensitivities to the peptide ligand. In other words, delay in the switch to ecdysis could result from a lower density of ETHR in CAMB neurons, making them less sensitive to ETH. Possible differential sensitivity to ETH could be tested in variety of way, including assessing timing of responses to the ligand by acutely dissociated neurons and/or single cell PCR (Kim, 2015).

A mechanistic model is proposed to explain neural mechanisms underlying the Drosophila pupal ecdysis FAP (see A model depicting functional roles of kinin and CAMB neurons in scheduling of the ecdysis FAP). Principle players in orchestration of pre-ecdysis and ecdysis behaviors are the kinin and CAMB ETHR ensembles, respectively. As ETH levels rise in the hemolymph, ETHR-B neurons are activated due to their high sensitivity (EC50 ~ 1 nM). These neurons release inhibitory signals acting through Gαi/o to inhibit CAMB neurons. As ETH levels rise further, kinin neurons receive direct excitatory input from ETH signaling via ETHR-A and Gαq to mobilize calcium from intracellular stores, leading to electrical activity in these neurons. ETH acts simultaneously on CAMB neurons, but inhibition from ETHR-B neurons descending from anterior ganglia prevents them from becoming active. As inhibition wanes, CAMB neurons become active, initiating the switch to ecdysis behavior (Kim, 2015). Antagonistic actions of ecdysone and of insulins, acting through Foxo and 4E-BP, determine final size in Drosophila

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).

In metazoans, the insulin/IGF signaling pathway (IIS) plays a key role in regulating growth and metabolism. In Drosophila, a family of insulin-like molecules called Dilps activates a unique insulin receptor (InR) and a conserved downstream kinase cascade that includes PI3-kinase (PI3K) and Akt/PKB. Recent genetic experiments have established that this pathway integrates extrinsic signals such as nutrition with the control of tissue growth during larval stages. The larval period is critical for the control of animal growth, since it establishes the size at which maturation occurs and, consequently, the final adult size. Maturation is itself a complex process that is controlled by the steroid 20-hydroxyecdysone (20E). Peaks of 20E determine the timing of all developmental transitions, from embryo to larva, larva to pupa, and pupa to adult. Ecdysteroids are mainly produced by the prothoracic gland (PG), a part of a composite endocrine tissue called the ring gland. Final adult size thus mainly depends on two parameters: the speed of growth (or growth rate), which is primarily controlled by IIS, and the overall duration of the growth period, which is limited by the onset of the larval-pupal transition and timed by peaks of ecdysone secretion. Very little is known concerning the mechanisms that coordinate these two parameters during larval development (Colombani, 2005).

To investigate the function of ecdysone in controlling organismal growth, a genetic approach was developed that allowed modulation basal levels of ecdysteroids in Drosophila. The initial rationale was to modify the mass of the ring gland in order to change the level of ecdysteroid production. For this goal, the levels of PI3- kinase activity were manipulated in the PG by crossing P0206-Gal4 (P0206>), a line with specific Gal4 expression in the PG and corpora allata (CA), with flies carrying UAS constructs allowing expression of either wild-type (PI3K) or dominant-negative (PI3KDN) PI3-kinase. As expected, these crosses produced dramatic autonomous growth effects in the ring gland, and particularly in the PG: tissue size was increased upon PI3K activation and decreased upon inhibition. Surprisingly, the changes in ring gland growth were accompanied by opposite effects at the organismal level. P0206>PI3K animals (with large ring glands) showed reduced growth at all stages of development and produced emerging adults with reduced size and body weight (78% of wt). Conversely, reducing PI3K activity in the ring gland of P0206>PI3KDN animals led to increased growth and produced adults with 17% greater weight on average. Adult size increase was attributable to an increase in cell number in the wing and the eye. Adult size reduction was accompanied by a decrease in cell number in the wing and in cell size in the eye (Colombani, 2005).

Importantly, the timing of embryonic and larval development of these animals was comparable to control. Both the L2 to L3 transition as well as the cessation of feeding (wandering) occurred at identical times. Further, animals entered pupal development at the same time, except for P0206>PI3KDN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3KDN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3KDN animals, albeit by less than 4 hours following 10 days of development. In contrast, the speed of larval growth was found to be increased in P0206>PI3KDN animals and decreased in the P0206>PI3K animals background at the earliest stage that could be measured (early L2 instar). Because none of these effects were observed when PI3K levels were modified specifically in the CA using the Aug21- Gal4 driver, it was concluded that the observed phenotypes are solely due to PI3K modulation in the PG. Together, these results demonstrate that manipulating PI3K levels in the PG induces non-autonomous changes in the speed of larval growth (growth rate effects), without changing the timing of larval development (Colombani, 2005).

To investigate whether these effects could be attributed to changes in 20E levels, ecdysteroid titers were measured in third instar larvae of the different genotypes. Early after ecdysis into third instar (74hrs AED) ecdysteroids are present at basal level. They accumulate to an intermediate plateau around 90hrs AED and reach peak levels before pupariation (120hrs AED). Because early L3 levels are below the detection limit of the EIA assay, ecdysteroid titers were measured at the intermediate plateau (90hrs AED). In these conditions, a very modest increase of ecdysteroids was observed in P0206>PI3K animals larvae and a small but significant decrease in P0206>PI3KDN animals animals. This was further confirmed by measuring the transcript levels of a direct target of 20E, E74B, which responds to low/moderate levels of 20E. However, in early L3 larvae with basal ecdysteroid levels (74hrs AED), differences in E74B transcripts were clearly visible, with a 1.9-fold increase seen for P0206>PI3K animals and a 1.7-fold decrease for P0206>PI3KDN animals. This establishes that basal circulating levels of 20E are modified in response to manipulation of PI3K levels in the PG. It also suggests that the differences observed on basal 20E level off with the strong global increase of ecdysteroids in mid/late L3 (Colombani, 2005).

Several related lines of evidence strengthen these results: (1) the increase in growth rate and size observed in P0206>PI3KDN animals can be efficiently reverted by adding 20E to their food; (2) feeding wild-type larvae 20E recapitulates the effects observed in P0206>PI3K animals animals; (3) ubiquitous silencing of EcR using an inducible EcR RNAi construct results in a growth increase similar to that observed in P0206>PI3KDN larvae. Finally, the phantom (phm) and disembodied (dib) genes, which are specifically expressed in the PG and encode hydroxylases required for ecdysteroid biosynthesis, showed 1.65- and 2.2- fold increased expression, respectively, upon PI3K activation in the ring gland. This supports the notion that 20E biosynthesis is mildly induced in these experimental conditions. In line with previous results, neither 20E treatment nor EcR silencing has any effect on developmental timing. Overall, the results indicate that manipulating basal levels of 20E signaling in various ways modifies the larval growth rate without affecting the timing of the larval transitions (Colombani, 2005).

Variations in ecdysone levels in animals with different sized ring glands could be due to changes in the PG tissue mass or, alternatively, to a specific effect of PI3K signaling in the secreting tissue. To distinguish between these two possibilities, extra growth was induced in the PG using either dMyc or CyclinD/Cdk4, two potent growth inducers in Drosophila. Although the size of the larval ring gland was markedly increased under these conditions, no effect on pupal or adult size was observed, implying that the size of the ring gland is not the critical factor in the control of body size. Instead, it is likely that the InR/PI3K signaling pathway can specifically activate ecdysone production from the PG (Colombani, 2005).

The possibility was tested that ecdysone signaling opposes the growth-promoting effects of IIS in the larva. To test this, larvae were fed 20E and xPI3K activity was assessed in vivo using a GFP-PH fusion (tGPH) as a marker. Membrane tGPH localization shows a marked decrease in the fat body of 20E-fed animals, and this effect can be reverted by specifically silencing EcR in the fat body. This indicates that ecdysone-induced growth inhibition correlates with decreased IIS, and is mediated through the nuclear receptor EcR. Conversely, larvae with PI3KDN expression in the PG show a 4.2-fold increase in the global levels of dPKB/Akt activity, as measured by the phosphorylation levels of serine 505. In Drosophila cells (as in other metazoan cells) high levels of PI3K/AKT activity cause the transcription factor dFOXO to be retained in the cytoplasm, while low PI3K/AKT activity allows dFOXO to enter the nucleus where it promotes 4E-BP transcription. In larvae with ectopic PI3K expression in the PG, a strong increase is observed in nuclear dFOXO in fat body cells. Similar results were obtained by feeding larvae with 20E. Conversely, inactivation of EcR signaling in fat body cells was carried out using the clonal over-expression of a dominant-negative form of EcR (EcRF645A). In these conditions, a reduction was observed in the accumulation of dFOXO in the nuclei of EcRF645A-expressing cells. As an expected consequence of the increased nuclear dFOXO, global accumulation of 4E-BP transcripts was consistently higher in P0206>PI3K animals as well as in 20E-fed early L3 larvae as compared to control animals, and reduced in arm>EcR-RNAi animals. Together, these results indicate that ecdysone-dependent inhibition of larval growth correlates with a general alteration of insulin/IGF signaling, and a relocalization of dFOXO into the cell nuclei. To more directly test the role of dFOXO in the growth-inhibitory function of ecdysone signaling, the effects of increasing ecdysone levels were examined in a dFOXOmutant genetic background. Although homozygous dFOXO21 animals do not display a detectable growth phenotype, introducing the dFOXO21 mutation was sufficient to totally revert the growth defects of P0206>PI3K animals animals, either when homozygous or heterozygous. This data establishes that dFOXO is required for 20E-mediated growth inhibition (Colombani, 2005).

The endocrine activities of the brain and the fat body have previously been implicated in the humoral control of larval growth. In order to test for possible roles of these two organs in mediating the systemic growth effects of ecdysone, EcR expression was silenced specifically in the brain cells that produce insulins (IPCs) or in the fat body. While specific expression of EcR RNAi in the IPCs fails to reproduce the overgrowth observed in armGal4>EcR-RNAi animals, EcR silencing in the fat body elicits an acceleration of larval growth and a remarkable increase in pupal size. Moreover, no detectable delay in the larval timing was observed in pplGal4>EcR-RNAi animals. Thus, specifically reducing 20E signaling in the fat body is sufficient to recapitulate the systemic effects of global EcR silencing. This demonstrates that the fat body is a major target for ecdysone, and that this tissue can act to relay the 20E growth-inhibitory signal to all larval tissues (Colombani, 2005).

In summary, these results establish an additional role for 20E in modulating animal growth rates. This function is mediated by an antagonistic interaction with IIS that ultimately targets dFOXO function. A similar antagonistic interaction between 20E and insulin signaling controls developmentally-regulated autophagy in Drosophila larva (Colombani, 2005).

Although a direct effect of ecdysone on the cellular growth rate of all larval tissues cannot be ruled out, the experiments reveal a key role for the fat body in relaying ecdysone-dependent growth control signals. Together with previous work, these data suggest that various inputs such as nutrition and ecdysone converge on this important regulatory organ, which then controls the general IIS to modulate organismal growth (Colombani, 2005).

How then is growth connected to developmental timing? The finding that 20E can modulate growth rates in addition to developmental transitions places this hormone in a central position for coordinating these two key processes and controlling organismal size (Colombani, 2005).

Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis

The fusion of epithelial sheets is an essential morphogenetic event. The development of the abdomen of Drosophila was studied as a model of bounded epithelia expansion; a complex multistep process was uncovered for the generation of the adult epidermis from histoblasts, founder cells that replace the larval cells during metamorphosis. Histoblasts experience a biphasic cell cycle and emit apical projections that direct their invasive planar intercalation in between larval cells. Coordinately, the larval cells extrude from the epithelia by apical constriction of an actomyosin ring and as a consequence die by apoptosis and are removed by circulating haemocytes. The proliferation of histoblasts and the death of larval cells are triggered by two independent extrinsic Ecdysone hormonal pulses. Histoblast spreading and the death of larval cells depend on a mutual exchange of signals and are non-autonomous processes (Ninov, 2007).

Ecdysone acts as a significant temporal signal in Drosophila, triggering each of the major developmental transitions. Although most of the genetic elements involved in Ecdysone signal transmission are known, the difficulty in visualizing morphogenetic changes in vivo and interfering with signal reception in individual cells has become a major impediment in understanding of Ecdysone actions during metamorphosis (Ninov, 2007).

In vitro culture studies have shown that Ecdysone pulses are crucial for the morphogenesis of adult appendages. Other studies have uncovered the ecdysteroid dependence of multiple differentiative and maturational responses. Nonetheless, little is known about ecdysteroid control of cell proliferation. A revealing analysis in Manduca showed that proliferating cells of the optic lobe reversibly arrest in G2 whenever the concentration of ecdysteroid drops below a critical threshold. Furthermore, earlier studies had shown that the number of histoblasts is reduced in hypomorph mutants for EcR isoforms. Whether this was the result of lack of proliferation or cell death had not been defined. This study shows in vivo a direct role for Ecdysone in cell proliferation. The rapid cell cycles experienced by abdominal histoblasts at the end of the third larval instar halt if Ecdysone-signalling reception is cell-autonomously compromised. Histoblasts remain quiescent in G2 and competent to resume proliferation in response to late Ecdysone pulses. These observations suggest that Ecdysone signalling controls the cell cycle by regulating the expression of genes involved in the G2-to-M transition (Ninov, 2007).

The destruction of larval tissues in Drosophila also results from a major transcriptional switch triggered by Ecdysone. The anterior larval muscles and larval midgut and the head and thoracic LECs degenerate during the first half of prepupal development (prepupal Ecdysone peak), while the larval salivary glands, abdominal muscles and abdominal polyploid larval epidermal cells (LECs) histolyze after the second Ecdysone pulse (pupation). Given that the exposure to Ecdysone is systemic, the stage-specific cell death responses of different cell types to Ecdysone must be differentially regulated (Ninov, 2007).

The death of abdominal LECs shows apoptotic characters and proceeds in two steps: the basal extrusion of cells initiated by the contraction of an apical actomyosin ring, and their removal by haemocytes. The cell-autonomous inhibition of EcR activity in LECs led to abortive extrusion and cell survival. Thus, the death of LECs share with other obsolete larval cells a common priming hormonal (Ecdysone) input (Ninov, 2007).

It is still not clear how cell proliferation and cell death are differentially controlled by Ecdysone. The trigger of histoblast proliferation seems to be directly dependent on Ecdysone signalling. However, it is still not known how the onset of LEC death is set. In other words, how do LECs distinguish between the late larval and the pupal Ecdysone pulses? In a plausible scenario, to avoid detrimental epithelial gaps at the surface, signalling clues from 'matured' histoblasts (after their rapid proliferation in response to the initial prepupal Ecdysone pulse), could assist Ecdysone signalling to instruct LECs to die. Indeed, LECs do not die in response to the pupal Ecdysone pulse if histoblast proliferation (and hence, 'maturation') has been experimentally delayed. The identification and characterization of this putative signal awaits further genetic and molecular analysis. Thus, Ecdysone signalling is necessary, but not sufficient, for LEC death (Ninov, 2007). The developmental control of cell cycle dynamics and diversity represents a key regulatory mechanism that directs cell size, cell number and ultimately the organ size of adult individuals. Despite numerous elegant experiments, the details of how cell division is regulated and coupled to cell growth remain poorly understood (Ninov, 2007).

During abdominal morphogenesis, the trigger of cell proliferation occurs simultaneously in all histoblast nests within each segment. Cell counting reveals that up to eight cell divisions are required to build the complete adult hemitergite. The same proportions apply to the ventral and spiracular nest. The first three histoblast divisions during pupariation are synchronous and extremely fast, skip the G1 phase and resemble the early embryonic blastoderm divisions. In this early stage, histoblast cleave and progressively reduce their size. Ecdysone signalling was found to be involved in the initiation of the proliferation programme. But, how is the histoblast cell cycle regulated to achieve fast proliferation in the absence of cell growth? Does it rely on the storage of preexistent control molecules, as in early embryos, or is it linked to signals impeding their growth? While this issue remains to be unravelled, the extreme growth of histoblasts during previous larval stages makes plausible the accumulation of G1 regulators, which, upon Ecdysone signalling, could allow a fast transition through G1 phase. Indeed, it was found that Cyclin E concentration (which regulates entry into S phase) in histoblasts builds up during the larval period. The observed deceleration of histoblast proliferation could then be the consequence of the exhaustion of the entire stock of Cyclin E. Still, the implication of growth control mechanisms in the regulation of histoblast proliferation cannot be ruled out. Multiple cell types, such as the animal-cap blastomeres from Xenopus embryos, change their cell cycles from size-independent to size-dependent after they become smaller than a critical cell size. Histoblasts might sense size in an analogous way. Thus, pathways that regulate growth, such as insulin-mediated signalling, Myc and Ras oncoproteins and the products of the Tuberous sclerosis complex 1 and 2 genes (reviewed by 22-122">Jorgensen and Tyers, 2004), should be explored to evaluate their potential roles in the coupling mechanism linking growth and cell cycle progression (Ninov, 2007).

Histoblast nest spreading initiates with the projection of leading comet-like protrusions, followed by apical cytoskeletal activity and active crawling over the underlying basal membrane, and terminates with the implementation of an apparent purse string, reminiscent of those described during dorsal closure, C. elegans ventral enclosure or wound healing (Ninov, 2007).

The comet-like protrusions of guiding histoblasts break through the LEC epithelial barrier, leading to planar intercalation of histoblast cell bodies. They account for the capacity of histoblasts to achieve migration within the bounded epithelial layer. Indeed, electron micrographs reveal that the advancing histoblasts form junctions with non-adjacent LECs before the adjacent LECs histolyze, thus insuring the continuity of the epidermis. Time-lapse observations suggest that these protrusions grow by sequential addition of actin molecules at their forward end. In this sense, they resemble, although being considerably slower, the actin tails employed by Listeria to propel through the cytoplasm of infected cells, or the actin-rich pseudopodia extended by neutrophils in response to chemoattractants. Proper actin cytoskeleton dynamics appear to be essential to build up these protrusions and the full repertoire of activities leading to the expansion of histoblast nests. The equilibrium between actin polymerization and depolymerization activities should be exquisitely regulated, and the forced polymerization of actin by Profilin overexpression not only blocks the cytoskeletal dynamics of single cells, but impedes the spreading of the whole histoblast nest. Potential roles for further actin dynamics regulators, the Arp2-Arp3 (Arp14D-Arp66B - FlyBase) complex, Dynamin (Shibire), membrane polyphosphoinositides, Cdc42, WASp-family proteins and other molecules in building up these projections remain to be explored. Further, although these protrusions appear to have a mechanical role, they also seem to be involved in the recognition of guidance cues, as they follow stereotyped paths. Indeed, gradients of cell affinity have been described for the patterning of the Drosophila abdomen, and it would be of major interest to understand how these cells interpret the larval landscape (Ninov, 2007).

The mechanisms involved in the death of LECs have been a matter of debate. While ultrastructural analysis suggests that LECs are phagocytosed, other studies suggested that LECs are histolyzed and die by autophagy. The current findings are conclusive in this respect. The death of LECs involves a caspase-mediated apoptotic process that implicates cytoskeletal remodelling and apical cellular constriction leading to delamination. The actomyosin mediated contractile force of dying LECs contributes in bringing together neighbouring histoblasts. Once the LECs initiate extrusion, they become immediate targets for circulating haemocytes, which extend membrane projections and engulf them. Finally, LECs are degraded inside haemocytes (Ninov, 2007).

Apical constriction is a process shared by multiple morphogenetic events, e.g., Drosophila mesodermal cells accumulate myosin and apically constrict during gastrulation under the control of the small GTPase Rho. Myosin activity is also sufficient to promote the apical constriction and elimination of photoreceptor cells in the Drosophila eye in response to the overexpression of an activated form of the Rok kinase. Indeed, this study found that the apical contractility of LECs depends on the level of phosphorylation of the MRLC and could be enhanced or abolished by modulating the counteracting kinase and phosphatase activities of Rok and MLCP. As a consequence, LEC delamination is either accelerated or delayed. How these regulatory activities are themselves regulated remains to be established. Yet, the LEC extrusion defects observed in weakened caspase cascade conditions after P35 overexpression strongly suggest that apoptotic signals could be involved in the trigger of actomyosin contractility in LECs. Apical contraction would thus be an early event in the LEC apoptotic process. Being particularly important to analyse the differences that modulate the activity of myosin during apical constriction of living cells and during extrusion of apoptotic cells, the replacement of LECs could become an exceptionally suitable model to unravel how myosin activity is regulated in apoptotic cells in vivo (Ninov, 2007).

The recruitment of haemocytes to dying LECs during abdominal cell replacement is extremely fast. The apical constriction of LECs takes about 2 hours, but the time that a haemocyte needs to fully engulf a LEC is less than 10 minutes. This entails a very reliable chemoattracting mechanism. In mammals, caspase 3-dependent lipid attraction signals, released by dying cells, induce the migration of phagocytes. Furthermore, several receptors are implicated in corpse recognition, including lectins, integrins, tyrosine kinases, the phosphatidylserine receptor (PSR) and scavenger receptors. In Drosophila, the elements involved in cell recognition by macrophages are mostly unknown. Haemocytes express Croquemort, a scavenger receptor homologue, which is required for the uptake of dead cells, and Pvr, a homologue of the vertebrate PDGF/VEGF receptor that seems to affect their motility. Still, the signals that haemocytes recognize in dying cells and the links between those signals and the apoptotic cascade are essentially unknown (Ninov, 2007).

As macrophages are responsible for much of the engulfment of dead cells in developing animals, an important role for macrophages in tissue morphogenesis has been suggested. However, this is not the case during abdominal morphogenesis, as the inhibition of haemocyte motility, which abrogates the removal of LECs, does not affect their replacement by histoblasts. These results are consistent with studies showing that macrophage removal of cell debris is not required for the regeneration of laser-induced wounds in Drosophila (Ninov, 2007).

Histoblast nest expansion is tightly coordinated with LEC removal. A naive view of the process of LEC extrusion suggests that their death is altruistic - it would promote the expansion of histoblasts. However, several results suggest that LECs do not execute this process autonomously. First, histoblast nests initiate their expansion in the absence of LEC death. Second, histoblast nests, during their spreading, grow, with no obvious planar orientation, by stochastic cell divisions not restricted to their edges. Finally, and most importantly, the inhibition of histoblast proliferation exerts non-autonomous effects on both extrusion and removal of LECs. A working model in which histoblast proliferation and LEC death are synchronized by a spatially and temporally controlled exchange of signals (secreted ligands or cell-to-cell communication modules) is thus strongly appealing. This potential mechanism for replacement of LECs by histoblasts somewhat resembles the elimination and death by anoikis of amnioserosa cells upon dorsal closure completion during Drosophila embryogenesis. Through this process, physical contacts and intracellular signalling among epithelial leading cells, the amnioserosa and the yolk sac coordinate the different behaviour of these cell types, which is essential for the accurate progress of both germ band retraction and dorsal closure. In this scenario, coordinated extrinsic and intrinsic events, hormonal inputs, cell contacts and cell signalling events will be responsible for the ordered proliferation and expansion of histoblasts and the extrusion and death of LECs (Ninov, 2007).

An alternative mechanism for the ordered cell substitution taking place during abdominal morphogenesis involving cell competition could also be proposed. Competition can be defined as an interaction between individuals brought about by a shared requirement leading to a reduction in the survivorship, growth and/or reproduction rates. Classical experiments in Drosophila imaginal discs have shown that cells heterozygous mutant for ribosomal protein genes (Minutes) placed beside wild-type cells are outcompeted and eliminated from the epithelium. More recent work has shown that imaginal wild-type cells are outcompeted by cells with growth advantage overexpressing the proto-oncogene Myc. Cell competition does not just apply to the fight for survival of cells with their 'fitness' experimentally altered, but also applies to the homeostasis of self-renewing cell pools such as lymphocytes or stem cells. The substitution of LECs by histoblasts closely resembles cell competition. Rapidly dividing and expanding histoblasts may become competent to displace the surrounding less-metabolically-active LECs. During normal development, having 'weaker' neighbours, histoblasts do not compete against each other, and cells from Minute clones in the abdomen are not eliminated in heterozygous animals. However, when confronted with death-resistant LECs, 'winner' histoblasts may become 'losers'. Histoblasts in an increasingly crowded environment will compete against each other, and the less fit individuals (less competent in signalling reception and transduction, or with slower proliferation rates) would eventually become more sensitive to 'killing' signals and would die (Ninov, 2007).

These findings demonstrate that the replacement of LECs by histoblasts, independently of being driven by cooperative mechanisms, cell competition or both, represents an extremely amenable morphogenetic model for the study of the dynamic control of the cell cycle and cell death, of the coordination of cytoskeleton activities and cell adhesion, and for the study of cell invasiveness (Ninov, 2007).

Interaction between Drosophila bZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. This study implicates Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. These data thus identify Atf3 as a new functional partner of Drosophila Jun during development (Sekyrova, 2010).

Metamorphosis of Drosophila larvae into pupae and adult flies provides remarkable examples of morphogenetic changes that involve replacement of entire cell populations. Epithelia that had served larval function undergo programmed cell death while imaginal cells proliferate and differentiate to take their position. The Drosophila abdomen is an attractive system for studying the developmental replacement of one epithelial cell population with another. Unlike the adult head and thorax with appendages, all forming from pre-patterned imaginal discs, the adult abdomen derives from histoblasts that reside in each abdominal segment. Soon after the onset of metamorphosis, the diploid histoblasts undergo an initial phase of synchronized cell divisions; later the histoblasts expand while proliferating and replace the old polyploid larval epidermal cells (LECs) that cover the surface of the abdomen. To free space for the histoblasts, LECs are extruded from the epithelial monolayer. In order to maintain integrity of the epithelia, changes in cell adhesion and cell migration must be precisely orchestrated during this tissue remodeling (Sekyrova, 2010).

Rho kinase signaling, which stimulates constriction of the apical actomyosin cytoskeleton through myosin phosphorylation, is necessary for the extrusion and the ensuing apoptosis of LECs. Perturbed myosin phosphorylation leaves the process of the epithelial exchange incomplete, with residual LECs obstructing closure of the adult abdominal epidermis at the dorsal midline. A similar defect results from compromised function of the ecdysone receptor (EcR), which is required for both the initial phase of histoblast proliferation and for the removal of LECs. Other factors besides Rho signaling and EcR that regulate the epithelial cell replacement are unknown (Sekyrova, 2010).

This study implicates Atf3 (A3-3 -- FlyBase), the single Drosophila ortholog of the vertebrate Activating transcription factor 3 (ATF3) and Jun dimerization protein 2 (JDP2) in abdominal development. ATF3 and JDP2 belong among basic region-leucine zipper (bZIP) proteins, some of which play important roles in epithelial morphogenesis. Particularly the functions of Jun and Fos bZIP proteins in epithelial closure events during development are well understood owing to genetic studies in Drosophila. By contrast, no morphogenetic function has yet been reported for Atf3 in Drosophila (Sekyrova, 2010).

Mammalian ATF3 and JDP2 form homodimers but preferentially dimerize with members of the Jun subfamily (Aronheim, 1997; Hai, 1989; Hsu, 1991), functioning either as transcriptional activators (ATF3-Jun) or repressors (JDP2-Jun). Based mainly on cell-culture studies, multiple roles in cell proliferation, differentiation and apoptosis have been ascribed to ATF3 and JDP2. Atf3-/- mice are viable but suffer from altered glucose and immune homeostasis. Also Jdp2-/- mice survive but produce extra fat in their brown adipose tissue. In vivo significance of the interaction between the ATF3 or JDP2 proteins and Jun remains unclear (Sekyrova, 2010).

This study shows that Atf3 interacts biochemically and genetically with Jun in Drosophila. Temporal downregulation of atf3 transcription during metamorphosis is crucial, since sustained atf3 expression alters adhesive properties of LECs, thus preventing their extrusion and replacement by the adult epidermis. This effect of Atf3 requires the presence of Jun (Sekyrova, 2010).

Among Drosophila bZIP proteins, the predicted product of the CG11405 gene (also referred to as a3-3), located on the X chromosome, shows the closest similarity to the mammalian ATF3 and JDP2 proteins. The DNA-binding/dimerization bZIP domains of the human ATF3 and Drosophila Atf3 proteins are identical in 60% of their amino acids; there is 58% identity between Atf3 and JDP2 in this region (Sekyrova, 2010).

Dimerization between Atf3 and Jun in Drosophila has been theoretically predicted and confirmed by a yeast two-hybrid screen. To demonstrate direct binding, co-immunoprecipitation experiments were conducted. The endogenous Jun protein from Drosophila S2 cells co-precipitates with a transiently expressed Atf3 whereas Fos did not. A DNA mobility-shift assay with recombinant bZIP domains of Atf3, Jun and Fos was conducted to test for their DNA-binding properties. Atf3 specifically bound an ATF/CRE consensus element but not the AP-1 site, which was recognized by the Jun-Fos (AP-1) complex. Although Atf3 bound DNA by itself, presumably as a homodimer, the binding was enhanced in the presence of Jun. Fos did not synergize with Atf3 in DNA binding. Excess unlabeled DNA bearing the ATF/CRE binding site competed for the Atf3 bandshift activity whereas the AP-1 binding element did not. These results have shown that, like ATF3 or JDP2 in mammals, Atf3 in Drosophila selectively dimerizes with Jun, with which it cooperatively and specifically binds the ATF/CRE DNA element (Sekyrova, 2010).

To test whether Atf3 and Jun interact in vivo, experiments were conducted in the Drosophila compound eye, the precise structure of which sensitively reflects genetic interactions. Overexpression of atf3 under the GMR-Gal4 driver disrupted the ommatidial arrangement, resulting in smaller eyes with a glossy appearance. This atf3 misexpression phenotype could be completely suppressed by simultaneous RNAi-mediated knockdown of jun but not of fos. Conversely, the phenotype was exacerbated when jun was overexpressed in the eye together with atf3, suggesting that it is Atf3 in a complex with Jun that derails the normal eye development. Neither RNAi nor overexpression of jun alone had any effect on eye morphology. Interestingly, like depletion of Jun, co-expression of fos under the GMR-Gal4 driver completely averted the atf3 misexpression phenotype, restoring the normal appearance of the eye. Expression of fos or its mutant versions alone had no effect. These data can be explained by the ability of the surplus Fos to bind Jun and thus reduce its availability for interaction with the Atf3 protein. This interpretation is further supported by experiments showing that expression of the truncated bZIP domain of Fos is sufficient to suppress the Atf3 gain-of-function phenotype, whereas its transcription activation domain or phosphorylation sites are dispensable (Sekyrova, 2010).

Taken together, these results show that Atf3 cooperates with Jun, as Jun is specifically required for an effect caused by overexpression of Atf3 in the developing eye. Given the capacity of both Atf3 and Fos to bind Jun, and based on the ability of Jun to enhance and of Fos to suppress the Atf3 gain-of-function phenotype, it is suggested that Atf3 and Fos compete for their common partner Jun in vivo (Sekyrova, 2010).

To find out whether Atf3 is required for Drosophila development and whether its absence might resemble a phenotype caused by loss of its partner Jun, atf3 mutant flies were generated. The longest deletion (line atf376) obtained by imprecise excision of a P element, removed the entire bZIP domain of Atf3, and atf376 hemizygous (male) larvae lacked detectable atf3 mRNA. Thus, atf376 probably represents a null allele. Most atf376 larvae die soon after hatching and during all three larval stages. Only a few (approximately 2%) reach the third instar but die before metamorphosis as defective pseudopuparia. Expression of atf3 cDNA under the ubiquitous armadillo (arm-Gal4) driver rescued some atf376 hemizygotes to adults, confirming that loss of atf3 was the cause of the lethal phenotype. Interestingly, the moribund atf376 larvae abnormally enlarged lipid droplets in their fat body, thus displaying a phenotype reminiscent of that in mice lacking one of the Atf3 orthologs, JDP2. However, in contrast to viable Jdp2 or Atf3 knockout mice, atf3 is an essential gene in Drosophila (Sekyrova, 2010).

Fly embryos lacking the function of Jun or Fos die because of the failed dorsal closure. However, atf376 embryos develop normally, without the dorsal open defect, even when derived from atf3-deficient germline clones induced in atf376/ovoD1 mothers. Thus, unlike its partner Jun, Atf3 is not required for dorsal closure, suggesting that dorsal closure is regulated by Jun-Fos dimers and that the Atf3-Jun complex has another function later in development (Sekyrova, 2010).

Consistent with the vital requirement for Atf3 during larval stages, atf3 mRNA was expressed in embryos and larvae. Expression then sharply declines by the late-third larval instar, and no atf3 mRNA was detected by northern blot hybridization in wandering larvae and during metamorphosis from the time of puparium formation until the second day of pupal development. Detailed RT-PCR analysis showed that atf3 downregulation coincided with the cessation of feeding and the onset of metamorphosis [0 hours after puparium formation (APF)]. A pulse of expression occurred at 6 hours APF. RT-PCR from isolated fat body and abdominal integuments, together with in situ hybridization performed on puparia at this stage, showed that atf3 mRNA was primarily present in the larval epidermis (LECs) during the expression peak at 6 hours APF. From the time of head eversion (12 hours APF) the mRNA level remained low until the second day of pupal development, and then it grew steadily during morphogenesis of the adult. Quantitative RT-PCR revealed a 4.3-fold difference in atf3 mRNA abundance between 0 and 72 hours APF. In contrast to the tight regulation of atf3, the mRNAs of fos and jun fluctuated little during the examined period. Therefore, unlike Jun or Fos, Atf3 was dynamically regulated during metamorphosis at the level of transcription (Sekyrova, 2010).

The precise temporal control of atf3 expression suggested that the rise and subsequent fall of Atf3 during metamorphosis might be critical for the complex morphogenesis occurring in fly pupae. This possibility was tested by means of sustained expression of the full-length Atf3 protein using the UAS-Gal4 system with various drivers. A striking, fully penetrant metamorphic defect was observed with the pumpless (ppl) Gal4 driver. Although ppl>atf3 animals developed normally until the pupal stage, they failed to complete fusion of the adult abdominal epidermis. A dorsal cleft in the abdomen remained that could not be covered with the adult cuticle, and consequently 86% of the flies died inside the puparium. All of the ppl>atf3 adults that did eclose showed abdominal lesions filled with the old pupal cuticle lacking adult pigmentation and bristles, often with a clot covering a bleeding wound. Adults with the same abdominal cleft (but otherwise normal) also emerged when atf3 was moderately and ubiquitously misexpressed under the arm-Gal4 driver, suggesting that abdominal morphogenesis was the process most sensitive to ectopic Atf3 (Sekyrova, 2010).

The adult fly abdomen derives from histoblasts that proliferate, replace LECs and finally differentiate, giving rise to the adult cuticle. Therefore, the observed abdominal defect suggested a compromised function of the epidermis, either LECs, histoblasts or both cell types. To distinguish between these possibilities, expression of the ppl-Gal4 driver was first examined in the epidermis. It was found that ppl-Gal4 was active in LECs but not in histoblasts. Second, another driver, Eip71CD-Gal4, which was inactive in histoblasts but strongly expressed in LECs, was examined. Eip71CD-Gal4-driven misexpression of atf3 mostly produced lethal pupae lacking adult cuticle, but it occasionally yielded adults with a dorsal abdominal cleft. In addition to being active in LECs, both ppl-Gal4 and Eip71CD-Gal4 (data not shown) were also expressed in the fat body. However, no abdominal defects occurred when atf3 was misexpressed under either of three fat-body-specific Gal4 drivers, Lsp2, Cg or C7. Third, to rule out the possibility that ectopic Atf3 affected the imaginal epidermis, its expression was directed to histoblasts by using the escargot (esg) and T155 Gal4 drivers; in neither case the fusion of the adult abdominal epidermis was affected (Sekyrova, 2010).

To finally confirm that abdominal morphogenesis was disrupted by sustained atf3 activity in LECs, atf3 was induced by using the flp-out technique. Owing to the timing of heat-shock induction to the mid-third instar, this method triggers expression in the polyploid larval cells but not in the diploid histoblasts . Misexpression of atf3 under the actin promoter following the flp-out event invariantly led to an abdominal cleft. The lesions were often more severe than those observed in ppl>atf3 animals, affecting also lateral and ventral parts of the abdomen. Together, the above data demonstrate that the sustained expression of atf3 prevents fusion of the adult abdominal epidermis by acting upon LECs, suggesting that the replacement of these obsolete larval cells by adult histoblasts requires the developmental downregulation of atf3 expression (Sekyrova, 2010).

To understand the cellular events underlying the incomplete epithelial closure in ppl>atf3 animals, cell membranes were visualized by antibody staining of the septate junction component, Discs large 1 (Dlg1), or used a transgenic DE-cadherin::GFP fusion protein (shg::gfp). In wild-type animals 24 hours APF, LECs covering the surface of the abdomen gave way to the rapidly expanding nests of histoblasts that began to fuse laterally and ventrally. In ppl>atf3 pupae the histoblast nests also spread, and at least at 16 hours APF, before their fusion, they comprised normal numbers of histoblasts. By 48 hours APF a control abdomen was fully covered with adult epidermis consisting exclusively of histoblasts, now forming sensory bristles. Histoblasts in ppl>atf3 abdomens also differentiated the adult cuticle with sensory bristles, although polarity of the bristles near the dorsal cleft was altered. However, in contrast to the control, a large population of LECs remained in the dorsal abdomen of ppl>atf3 animals at 48 hours APF. The membranes of the persisting LECs accumulated the Dlg protein, and although these cells became severely deformed they survived throughout metamorphosis to the adult stage. When visualized in live ppl>atf3 pupae, the apical junctions of the remaining LECs displayed interdigitation and accumulation of DE-cadherin::GFP. Another adherens junction component, the Drosophila β-catenin Armadillo, was also enriched in atf3-expressing LECs (Sekyrova, 2010).

Cooperation between adherens junctions and the apical ring of actomyosin cytoskeleton is required for basal extrusion of LECs. The altered pattern of DE-cadherin and β-catenin therefore suggests that excessive Atf3 might prevent LEC extrusion through stabilization of the cell-cell adhesion complex. To examine the effect of Atf3 on LECs in further detail, the flp-out technique, which allows comparisons of atf3-misexpressing and control LECs within one tissue, was employed. Membrane interdigitation occurred between atf3-positive LECs already at 18 and 24 hours APF, even in areas where the LECs had no contact with histoblasts. At 48 hours APF only LECs expressing atf3 persisted, apparently being squeezed by the expanding histoblasts. The membrane-associated DE-cadherin::GFP signal was stronger in adjacent atf3-positive LECs compared with non-induced LECs, and quantitative analysis of confocal images acquired at 18 hours APF and at 24 hours APF both revealed a statistically significant 1.4-fold increase of the DE-cadherin::GFP signal intensity upon atf3 induction. Enrichment of DE-cadherin on apical membranes of atf3-expressing LECs was further confirmed on confocal cross sections (Sekyrova, 2010).

Although some atf3-positive LECs began the extrusion process, they could not detach from the apical surface even when entirely surrounded by histoblasts, possibly being tethered to it by the excessive adhesion protein. By contrast, control LECs did completely separate from the epithelium. In addition, LECs overexpressing atf3 displayed apical enrichment of moesin, an actin-binding protein of the ERM (ezrin, radixin, moesin) family, which links transmembrane proteins to cortical actin filaments. Interestingly, prominent accumulation of DE-cadherin was also observed in atf3-expressing clones of epithelial cells within the hinge region of wing discs that form the adult thorax, indicating that the effect of Atf3 on cell adhesion components may not be limited to larval epithelia (Sekyrova, 2010).

In summary, these results show that deregulation of atf3 expression causes marked changes of cell membranes, including interdigitation and accumulation of cell adhesion molecules, suggesting that LEC adhesiveness might be increased. Although some of the affected LECs initiate extrusion, this process stays incomplete. Consequently, the adhering LECs present a physical barrier for the migrating histoblasts (Sekyrova, 2010).

Rho kinase (Rok)-dependent phosphorylation of myosin regulatory light chain was shown to be required for LEC extrusion. To examine a possible relationship between the Rok-dependent cytoskeletal regulation and Atf3, the function of the GTPase Rho1 (also called RhoA), which acts immediately upstream of Rok, was disrupted. RNAi silencing of Rho1 using the ppl-Gal4 driver produced a phenocopy of atf3 misexpression, causing a dorsal abdominal cleft in 100% of ppl>Rho1(RNAi) adults, of which most died in the puparium and about 12% eclosed, similar to ppl>atf3 animals. However, when Rho1 RNAi and misexpression of atf3 in LECs were combined, the abdominal defect became more severe, not allowing any pharate adults to eclose. Conversely, co-expression of a dominantly active Rho1V14 protein suppressed the otherwise fully penetrant abdominal defect in some ppl-atf3 flies. Surprisingly, it was found that the endogenous Rho1 protein was mislocalized in atf3-misexpressing LECs, showing a diffuse cytoplasmic signal, compared with membrane localization in control LECs. These results suggest a genetic interaction between Rho signaling and atf3, and support the idea that excess Atf3 prevents extrusion of LECs by altering their cell adhesion properties (Sekyrova, 2010).

Disturbed function of the ecdysone receptor (EcR) has been shown to prevent extrusion of LECs, causing a dorsal abdominal cleft that closely resembles the Atf3 gain-of-function phenotype. Therefore whether stimulating EcR-dependent signaling by addition of the natural agonist 20E might overcome the defect caused by sustained atf3 expression was examined. Indeed, supplying third-instar ppl>atf3 larvae with dietary 20E increased the number of eclosing adults, the abdominal scars of which were in 22% of the cases partially or completely sealed with normal adult cuticle (Sekyrova, 2010).

Atf3 interacts with Jun to form a DNA-binding complex and genetically when overexpressed in the developing compound eye. To see if this interaction is biologically relevant during abdominal morphogenesis, whether Atf3 relies on the presence of Jun to cause the dorsal cleft phenotype was tested. First, it was confirmed that Jun is indeed expressed in LECs during metamorphosis. RNAi-mediated depletion of Jun in animals that misexpressed atf3 under the ppl-Gal4 driver restored viability of adults from 14% (atf3 alone) to 100%. Strikingly, 87% of the ppl>atf3, jun(RNAi) adults eclosed with a completely normal abdomen. By contrast, RNAi knockdown of Fos in ppl>atf3 background did not improve the abdominal defect. RNAi silencing of either jun or fos alone under the ppl-Gal4 driver had no effect on the abdomen. These results demonstrate that Atf3 requires its partner Jun but not Fos to disrupt abdominal morphogenesis. Similar to the situation in the compound eye, the effect of misexpressed atf3 can be neutralized by simultaneously expressing Fos or its truncated bZIP domain under the ppl-Gal4 driver. Therefore, the model in which Atf3 and Fos compete for their common partner Jun may be extended to the developing abdomen (Sekyrova, 2010).

This study has identified Atf3 as a new partner of Jun in Drosophila. Previously, Jun has only been known to dimerize with itself and with the Drosophila homolog of Fos. Functional analysis of Atf3 has not yet been reported. These biochemical data show that, similar to mammalian ATF3 and JDP2, the Atf3 protein selectively binds Jun but not Fos. Also consistent with the properties of ATF3 and JDP2 is the ability of Atf3 to bind the ATF/CRE response element alone or synergistically with Jun. In contrast to its mammalian counterparts, however, neither Atf3 alone nor in complex with Jun bound to the AP-1 element under the same conditions. The selective interactions of Atf3 point to distinct biological roles for the Atf3-Jun and the Fos-Jun dimers, respectively (Sekyrova, 2010).

This study has shown a genetic interaction between Atf3 and Jun. The evidence is based on the ability of ectopic Atf3 to disturb morphogenesis of the adult abdomen and the compound eye, which strictly depends on the availability of Jun. Importantly, none of the Atf3 gain-of-function phenotypes could be induced by misexpression of the truncated bZIP domain of Atf3, suggesting that the functional Atf3 protein in complex with Jun is required. Based on the selectivity of Atf3 in a DNA-binding assay, it is predicted that the Atf3-Jun complex regulates specific target genes distinct from those targeted by Fos-Jun dimers (Sekyrova, 2010).

The data also reflect a relationship between the AP-1 and Atf3-Jun complexes. Although Fos does not dimerize or bind DNA with Atf3, its ability to suppress the Atf3 misexpression phenotype in the eye suggests that Fos and Atf3 compete in vivo for their common partner Jun. The fact that the same suppression can be achieved by overexpressing either the truncated Fos bZIP domain or Fos lacking phosphorylation sites indicates that the suppression does not rely on a transcriptional function of Fos but probably occurs through sequestering of Jun, even by a transcriptionally inactive Fos protein. Early in vitro studies have proposed a competition model for the AP-1 and Atf3 proteins to explain a temporal regulation of gene expression in the regenerating liver. However, to date such a relationship among Fos, Jun and Atf3 has not been supported with direct genetic evidence (Sekyrova, 2010).

Removal of LECs is normally complete by 36 hours APF, at which time the sheets of histoblasts reach the dorsal midline. The data strongly support the argument that the temporal downregulation of atf3 expression during abdominal morphogenesis is necessary for LECs to be replaced by the adult epidermis. When experimentally sustained, atf3 activity in LECs interfered with this exchange by blocking extrusion and death of the LECs. This was evident as the atf3-expressing LECs survived within the epithelial layer for days after their scheduled destruction (Sekyrova, 2010).

Interdigitation of cell membranes and accumulation of adherens junction proteins in LECs suggested that ectopic Atf3 caused adjacent LECs to reinforce their mutual contacts. This probably resulted from altered distribution of the proteins, as levels of the shg (DE-cadherin) mRNA remained unchanged in LECs of ppl>atf3 animals. By contrast, junctions between atf3-expressing LECs and their normal neighbors or histoblasts were smooth and presumably less rigid. DE-cadherin was similarly enriched in clones of imaginal disc cells. These observations suggested that differential adhesion of atf3-expressing cells might have led to their sorting out from the surrounding epithelium. Even modest differences in cadherin levels have been shown to cause segregation of cells within a population by altering their adhesiveness (Sekyrova, 2010).

Recent live imaging data have revealed that migrating histoblasts push the LECs ahead of themselves towards the dorsal midline, where histoblasts fuse last. The atf3-expressing LECs that adhered to each other were probably moved and pressed by the expanding histoblasts to the dorsal side, whereas non-induced LECs were eliminated. This explains why the abdominal lesions primarily occurred at the dorsal midline, although flp-out experiments showed that atf3 misexpression could affect LECs in other areas as well. Strengthened contacts among persisting LECs probably blocked invasion of histoblasts in between them and inhibited LEC extrusion, eventually causing gaps in the adult epidermis (Sekyrova, 2010).

In accord with the notion that extrusion from the epithelium is a prerequisite for LECs to undergo apoptosis, it is assumed that sustained presence of Atf3 primarily enhanced adhesiveness of LECs, which only consequently prevented their death. This view is supported by the observation that membranes of atf3-expressing LECs interdigitated and accumulated DE-cadherin as early as 18-24 hours APF, even in areas of the larval epidermis that were far from histoblasts and where control LECs did not yet extrude. In addition, the Atf3 gain-of-function phenotype was stronger than abdominal closure defects caused by caspase mutation or inhibition. When the anti-apoptotic proteins p35 or DIAP1 (Thread — FlyBase) was misexpressed under the ppl-Gal4 driver, the resulting dorsal lesions were not lethal and were clearly milder than the broad, mostly fatal scars in ppl>atf3 animals. Compared with the large contiguous populations of persisting LECs in ppl>atf3 pupae, inhibiting apoptosis with p35 only allowed small islands of LECs to survive (Sekyrova, 2010).

Ecdysone signaling promotes replacement of the abdominal epithelia by stimulating both the early histoblast proliferation and the extrusion of LECs. As atf3 misexpression affected LECs but did not impair early histoblast proliferation, the latter possibility remains, that added 20E counteracted the effect of ectopic Atf3 by facilitating the extrusion process. Since normal 20E titers was detected in ppl>atf3 larvae or prepupae, the failure of LEC extrusion was not a result of steroid deficiency. Also, 20E had no effect on atf3 mRNA levels, at least in Drosophila S2 cells or third-instar larvae. Atf3 and ecdysone signaling therefore probably influence LEC extrusion by acting independently (Sekyrova, 2010).

Although the mechanism through which ecdysone contributes to LEC removal is unknown, one attractive possibility is that it might cooperate with Rho signaling, which is required for LEC extrusion as well. It has been demonstrated that genetic interaction between the 20E-response gene broad and components of the Rho pathway including RhoGEF2, Rho1 and myosin II is important for ecdysone-dependent epithelial cell elongation during Drosophila leg morphogenesis. The current data show that Rho1 becomes mislocalized in LECs upon atf3 misexpression and that Rho1 silencing enhances the abdominal gain-of-function phenotype of atf3. The exact relationship between Atf3, Rho1 and ecdysone remains to be determined. However, Atf3 clearly represents a new intrinsic regulator of epithelial cell replacement during Drosophila metamorphosis (Sekyrova, 2010).

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) results in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), are significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, this study demonstrates that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

The status quo action of JH has been well documented in several insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, in which JH treatment causes supernumerary larval molting and JH deficiency triggers precocious metamorphosis. However, as JH does not cause supernumerary larval molting in flies, evidence for the status quo action of JH in Drosophila has remained elusive. From past studies and from the experimental data presented in this study, it is concluded that the status quo hypothesis does indeed apply to JH action in Drosophila. First, although JH application during the final larval instar or during the prepupal stage has little effect on the differentiation of adult head and thoracic epidermis in Drosophila, it does prevent normal adult differentiation of the abdominal epidermis. After JH treatment, a second pupal, rather than an adult, abdominal cuticle is formed in Diptera. Second, JH or a JH agonist applied to Drosophila at the onset of metamorphosis results in lethality during pupal-adult metamorphosis. Similarly, global overexpression of jhamt (Juvenile hormone acid methyl transferase) results in severe defects during the pupal-adult transition and eventually death (Niwa, 2008). Third, CA ablation leading to JH deficiency causes precocious and enhanced fat body PCD. Fourth, JH deficiency results in pupal lethality and delayed larval development, although JH deficiency is not sufficient to cause precocious metamorphosis. The composite data demonstrate that JH in Drosophila does have status quo actions on the abdominal epidermis during pupal-adult metamorphosis and on the fat body during larval-pupal metamorphosis. It is concluded that the status quo action of JH in Drosophila is functionally important, but more subtle than that in Coleoptera, Orthoptera and Lepidoptera. However, it is not clear whether JH is essential for embryonic and earlier larval development because the CA cells are not completely ablated in the JH-deficient animals until the early-wandering (EW) stage. To address this question, it would be necessary to generate a mutant (i.e., of jhamt) that interrupts JH but not the farnesyl pyrophosphate biosynthesis pathway (Liu, 2009).

The insect fat body is analogous to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. In response to 20E pulses, Drosophila larval organs undergo a developmental remodeling process during metamorphosis. Blocking the 20E signal specifically in the fat body during the larval-pupal transition (Lsp2>; UAS-EcRDN) prevented the fat body from undergoing PCD and cell dissociation (Liu, 2009).

The experimental data in this paper demonstrates that JH prevents caspase-dependent PCD in the fat body during the larval-pupal transition in Drosophila. First, JH deficiency in Aug21>, UAS-grim resulted in the fat body undergoing precocious and enhanced PCD and cell dissociation. Aug21> is a GAL4 driver that specifically targets gene expression to the CA. Precocious and enhanced apoptosis appeared as early as L3D1 in the JH-deficient animals. Methoprene application on L3D1 was able to rescue ~40% of the pupae to adults, but it failed to rescue post-EW. Second, 2D-DIGE/MS and qPCR analyses indicated that the fat body in the JH-deficient animals has multiple developmental defects. The upregulation of the caspase genes Dronc and Drice should account for the PCD in the fat body, as overexpression of Dronc in the fat body causes PCD, cell dissociation, and thus lethality. Overexpression of Dronc or Drice in cells and tissues is sufficient to cause caspase-dependent PCD. Third, the 20E-triggered transcriptional cascade in the fat body was downregulated in the JH-deficient animals, indicating that JH does not suppress the 20E-triggered transcriptional cascade in preventing caspase-dependent PCD in the fat body (Liu, 2009).

The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of EcR. One might expect that perfect timing, titer and receptor response of JH and 20E are required to ensure accurate PCD in a tissue- and stage-specific manner during Drosophila metamorphosis. In the JH-deficient animals, the upregulation of Dronc and Drice resulted in precocious and enhanced PCD, such that the JH-deficient animals are committed to die during the larval-pupal transition. This hypothesis was strengthened by overexpression of Dronc specifically in the fat body, which caused larval lethality. Taken together, it is concluded that JH antagonizes 20E-induced caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila (Liu, 2009).

Based on the phenotypes and gene expression profiles in the four fly lines used, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD. First, the Met-overexpressing animals died during larval life, with precocious and enhanced PCD and cell dissociation in the fat body. Dramatic upregulation of Dronc and Drice was observed when Met was specifically overexpressed in the fat body and this upregulation was significantly decreased by methoprene application demonstrating that JH is epistatic to MET and GCE. Moreover, the Dronc-overexpressing animals exhibited similar phenotypes to the Met-overexpressing animals. Second, in the fat body of the JH-deficient animals, PCD and the expression of Dronc and Drice were upregulated but not as significantly as in the Met-overexpressing animals. This might explain why the JH-deficient animals did not die until early pupal life. Third, both the global JH-overexpressing animals and the Met/gce-deficient animals died during the pupal-adult transition. In these animals, Dronc and Drice were downregulated and caspase-dependent PCD was decreased in the fat body, implying that these animals died from a lack of caspase-dependent PCD. Weak mutants of Dronc and Drice mutants die during pupal life, showing that caspase-dependent PCD is essential for Drosophila metamorphosis. In addition, it was also observed that methoprene application at the onset of metamorphosis results in delayed fat body remodeling (Liu, 2009).

In the future, it will be crucial to elucidate the detailed molecular mechanism of how JH counteracts MET and GCE to prevent caspase-dependent PCD. In Drosophila S2 cells, the transcriptional activity of MET is dependent on the JH concentration and both MET-MET and MET-GCE interactions can be greatly diminished by JH. The bHLH-PAS transcription factors typically function as hetero- or homodimers. If MET/GCE is the Juvenile Hormone Receptor (JHR), the transcriptional activities of the dimerized MET/GCE and the JH-MET/GCE complex should differ. In other words, the dimerized MET/GCE should induce transcription of Dronc and Drice and, in turn, JH binding to form the JH-MET/GCE complex should reduce this induction. Although there are no examples in the literature in which a receptor, without ligand, acts as a transcriptional activator and the transcriptional activity of the receptor is diminished when the ligand is bound, it could be speculated that the JHR is a unique hormone receptor and perhaps that is the reason why it has yet to be isolated and characterized. Unfortunately, the experiments described here were conducted in Drosophila S2 cells, where the possibility of an endogenous JHR could not be eliminated. Although MET/GCE is definitely a key component in the JH signal transduction pathway, whether MET/GCE is the bona fide JHR remains conjecture (Liu, 2009).

It is very likely that MET cross-talks with EcR-USP via a large molecular complex. One can hypothesize that MET promotes 20E action in the absence of JH and suppresses 20E action in the presence of JH, a model which is favored. Drosophila FKBP39 (FK506-BP1) could be a key component in this complex because it physically interacts with MET, EcR and USP, and binds the D. melanogaster JH response element 1. Moreover, Drosophila FKBP39 inhibits 20E-induced autophagy (Juhász., 2007). Further analysis of the complex will be crucial to precisely define the molecular mechanism of cross-talk between the action of JH and 20E (Liu, 2009).

In summary, it is concluded that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila. The Drosophila fat body has provided an excellent model for studying the long-standing question of JH signal transduction. To finally settle the question of the bona fide JHR and to understand the precisely defined molecular mechanism of JH action requires further research at a variety of levels in several species of insects that can be genetically manipulated, such as Drosophila, Bombyx and Tribolium (Liu, 2009).

DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase

Juvenile hormone (JH) biosynthesis in the corpus allatum (CA) is regulated by neuropeptides and neurotransmitters produced in the brain. However, little is known about how these neural signals induce changes in JH biosynthesis. This study reports a novel function of TGFβ signaling in transferring brain signals into transcriptional changes of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis. A Drosophila genetic screen identified that Tkv and Mad are required for JH-mediated suppression of broad (br) expression in young larvae. Further investigation demonstrated that TGFβ signaling stimulates JH biosynthesis by upregulating jhamt expression. Moreover, dpp hypomorphic mutants also induces precocious br expression. The pupal lethality of these dpp mutants is partially rescued by an exogenous JH agonist. Finally, dpp is specifically expressed in the CA cells of ring glands, and its expression profile in the CA correlates with that of jhamt and matched JH levels in the hemolymph. Reduced dpp expression was detected in larvae mutant for Nmdar1, a CA-expressed glutamate receptor. Taken together, it is concluded that the neurotransmitter glutamate promotes dpp expression in the CA, which stimulates JH biosynthesis through Tkv and Mad by upregulating jhamt transcription at the early larval stages to prevent premature metamorphosis (Huang, 2011).

The functions of the TGFβ superfamily and other morphogens in regulating insect metamorphosis are rarely reported. In two independent genetic screens, it was discovered that Drosophila TGFβ signaling controls two different aspects of insect metamorphosis. In a previous study, it was found that Baboon (Babo) and dSmad2-mediated TGFβ signaling regulates larval neuron remodeling, which is part of the insect central nervous system metamorphosis induced by 20E during the pupal stage. Further investigation revealed that Babo/dSmad2-mediated TGFβ signaling controls larval neuron remodeling through regulating the expression of EcR-B1, a specific isoform of the 20E receptor (Huang, 2011).

This paper reports several findings. First, br is precociously expressed in 2nd instar tkv and Mad mutant larvae. Second, the precocious br expression phenotype in tkv and Mad mutant larvae can be suppressed by exogenous JH agonist (JHA). Third, Tkv and Mad repressed br expression in a non-cell-autonomous manner. Fourth, the presence of Mad in the CA is sufficient to repress br expression in the fat body (FB). Fifth, jhamt mRNA levels and JHAMT activity were significantly reduced in the Mad-deficient larvae. These results demonstrate that Tkv- and Mad-mediated signaling is required in the CA to activate jhamt expression and thus JH biosynthesis, which in turn controls insect metamorphosis (Huang, 2011).

The Drosophila genome encodes two TGFβ type II receptors, Punt (Put) and Wishful thinking (Wit). The genetic screen failed to identify a role for either of these receptors in the regulation of JH biosynthesis. Put and Wit are most probably functionally redundant in this biological event, as in the case of TGFβ-mediated mushroom body neuron remodeling (Huang, 2011).

Dpp is a key morphogen that controls dorsal/ventral polarity, segmental compartment determination and imaginal disc patterning. Dpp function usually depends on its gradient distribution. In an attempt to identify the ligand for Tkv/Mad-mediated TGFβ signaling in the CA, a novel, gradient-independent role for Dpp was discovered that controls JH biosynthesis. Dpp is the ligand of Tkv, which regulates jhamt transcription. Loss of Dpp, even RNAi reduction of Dpp in the CA specifically, causes precocious br expression at the early larval stages, which phenocopies tkv and Mad mutants. Phenotypes of dpp, including precocious br expression and lethality, are at least partially rescued by JHA treatment or ectopic jhamt expression in the CA. Notably, dpp-lacZ is strictly expressed in the CA cells, but not in the other two types of endocrine cells in the ring gland: the prothoracic gland and corpus cardiacum cells. The developmental expression profile of dpp in the CA is always consistent with that of jhamt. Finally, dpp expression in the CA may be directly controlled by neurotransmitter signals in the brain, which is supported by reduced dpp and jhamt transcription levels in the Nmdar1 mutant wandering larvae (Huang, 2011).

Several lines of evidence suggest that Met is a crucial regulator at or near the top of a JH signaling hierarchy, possibly acting as a JH receptor. However, null Met mutants of Drosophila are completely viable, which is unexpected if Met is a JH receptor. A recent investigation indicated that another Drosophila bHLH-PAS protein, Germ cell-expressed (Gce), which has more than 50% homology to Met, may function redundantly to Met in transducing JH signaling (Baumann, 2010). Because Met is on the X chromosome in the fly genome, it was not covered by the genetic screen. The br protein in the FBs of a Met null allele, Met27, was tested at the 2nd instar larval stage, and precocious br expression was observed. Importantly, this precocious br expression phenotype could not be suppressed by exogenous JHA. This result not only supports the previous reports regarding the function of Met in transducing JH signaling but also suggests that the precocious br expression is a more sensitive indicator for the reduced JH activity in Drosophila compared with precocious metamorphosis, lethality and other phenotypes (Huang, 2011).

Kr-h1 was reported to act downstream of Met in mediating JH action. Studies in both Drosophila and Tribolium reveal that, at the pupal stages, exogenous JHA induces Kr-h1 expression, which in turn upregulates br expression. The genetic screen successfully identified that Kr-h1 is cell-autonomously required for the suppression of br expression at young larval stages. Precocious br expression occurred in the FBs of Kr-h1 mutants and was not suppressed by JHA treatment. Therefore, these studies further suggest that Kr-h1 functions as a JH signaling component in mediating insect metamorphosis. However, the finding shows that, at the larval stages of Drosophila, the JH-induced Kr-h1 suppresses, rather than stimulates, br expression. This result is consistent with the facts that Kr-h1 functions to prevent Tribolium metamorphosis and Br is a crucial factor in promoting pupa formation (Huang, 2011).

In summary, this study has found a novel function of Dpp, Tkv and Mad-mediated TGFβ signaling in controlling insect metamorphosis. As summarized in a model, the brain sends neurotransmitters, such as glutamate, to the CA through neuronal axons. Glutamate interacts with its receptor (NMDAR) on the surface of CA cells to induce dpp expression. Dpp protein produced and secreted by CA cells forms a complex with TGFβ type I receptor (Tkv) and type II receptor on the membrane of CA cells, followed by phosphorylation and activation of Tkv. Activated Tkv in turn phosphorylates Mad, which is imported into the nucleus together with co-Smad and stimulates jhamt expression. JHAMT in CA cells transforms JH acid into JH, which is released into hemolymph. The presence of JH in young larvae prevents premature metamorphosis through Met/Gce and Kr-h1 by suppressing the expression of br, a crucial gene in initiating insect metamorphosis (Huang, 2011).

bantam miRNA promotes systemic growth by connecting insulin signaling and ecdysone production

During the development of multicellular organisms, body growth is controlled at the scale of the organism by the activity of long-range signaling molecules, mostly hormones. These systemic factors coordinate growth between developing tissues and act as relays to adjust body growth in response to environmental changes. In target organs, long-range signals act in concert with tissue-autonomous ones to regulate the final size of a given tissue. In Drosophila, the steroid hormone ecdysone plays a dual role: peaks of secretion promote developmental transitions and maturation, while basal production negatively controls the speed of growth. The antagonistic action of ecdysone and the conserved insulin/insulin growth factor (IGF) signaling pathway regulate systemic growth and modulate final body size. This study has unraveled an unexpected role of bantam microRNA in controlling body size in Drosophila. The data reveals that, in addition to its well-characterized function in autonomously inducing tissue growth, bantam activity in ecdysone-producing cells promotes systemic growth by repressing ecdysone release. Evidence is provided that the regulation of ecdysone production by insulin signaling relies on the repression of bantam activity. These results identify a molecular mechanism that underlies the crosstalk between these two hormones and add a new layer of complexity to the well-characterized role of bantam in growth control (Boulan, 2013).

Because ban and ecdysone affect systemic growth in an opposite manner, it is likely that ban acts in PG cells by preventing ecdysone production. The circulating levels of the active form of ecdysone (20E) were measured in in P0206>ban animals at two developmental time points: at the beginning of the wandering phase (early L3w), and just before the larva/ pupa transition (late L3w). To do so, wandering larvae were precisely staged by monitoring gut clearance of blue food. As expected, 20E levels were already high in early L3w (blue gut) control larvae and further peaked in late L3w (clear gut). In contrast, P0206>ban larvae showed lower circulating 20E levels than controls at both stages, and the amplitude of the peak was strongly reduced. Ecdysone signaling in target tissues was also reduced in late larval development upon ban overexpression in the PG (in phm>banD animals) as measured by the expression levels of E75A and Broad-Complex (BR-C), two targets of EcR. Furthermore, the phantom (phm), disembodied (dib), and shade (sad) genes, which are specifically expressed in the PG and encode enzymes required for ecdysteroid biosynthesis, showed reduced expression in phm>ban animals. Consistent with the reduced levels of 20E production and signaling in phm>ban larvae, an increase in 20E levels, produced by feeding the animals ecdysone- supplemented medium, rescued pupa formation. These findings indicate that targeted expression of ban in ecdysone-producing cells has a negative impact on 20E production (Boulan, 2013).

banΔ1 mutant larvae displayed high lethality in late larval development. Thus, ecdysone signaling was assessed in these larvae during earlier stages. Two different developmental points precisely staged with respect to the transition from second (L2) to third (L3) larval instar were selected: 2 hr and 20 hr after ecdysis to the third instar (AL3E). No changes in the expression of BR-C were detected, most probably because at that time, 20E levels had not yet reached the minimum threshold to activate this target. However, the quantification of E75A mRNA levels revealed higher expression in banΔ1 larvae when compared to controls, as did the quantification of dib, phm, and sad mRNA levels. Ni predicted ban target site was found in the 3' UTR of these genes, suggesting that this repression is not direct (Boulan, 2013).

Ecdysone signaling negatively regulates body growth. To test whether the undergrowth phenotype observed in ban mutants is a result of abnormally high ecdysone levels, whole-mount ecdysone signaling was reduced by removing one copy of EcR or impaired ecdysone synthesis by depleting the levels of Sad and Phm in the PG of banΔ1 animals. In all cases, the size of banΔ1 pupae was largely rescued. Collectively, these results suggest that ban participates in reducing ecdysone production in PG cells and corroborate the hypothesis that the systemic growth defects observed in ban mutants are caused by increased ecdysone levels. Consistent with the cell-autonomous growth-promoting role of ban, the PG was larger in P0206>ban animals (that produce less ecdysone) than in controls, whereas it was much smaller in ban mutants. Thus, the impact of ban on ecdysone production is not a consequence of changes in PG size (Boulan, 2013).

Despite displaying higher levels of ecdysone, banΔ1 mutant animals reached metamorphosis with a delay. It has been reported that a strong reduction in larval growth rates can affect developmental timing as a result of a delay in the attainment of critical size for metamorphosis. In order to address whether banΔ1 animals are delayed as a consequence of their reduced growth rates, as a simple proxy of critical size, the time at which the minimal viable size for metamorphosis was achieved in was determined banΔ1 mutant and wild-type animals. Larvae were synchronized at the second (L2) to third (L3) instar transition and then starved at fixed time points to assess survival and capacity to enter into metamorphosis. Remarkably, banΔ1 larvae reached the threshold of 50% of survival with a delay when compared to wild-type animals. These data, together with the fact that targeted expression of ban in the ring gland largely rescued growth rates and developmental delay of banΔ1 animals, support the proposal that the developmental delay is at least in part a consequence of reduced growth rates. Other activities of ban, such as reduced growth of the imaginal tissues or impaired dendrite development, might also affect the timing of metamorphosis (Boulan, 2013).

The production of ecdysone is tightly controlled during larval development. Under normal conditions, ecdysone levels are low during the growth period, thereby allowing optimal body growth rates, and peak at the end of the third-instar larval stage to induce entry into metamorphosis. To monitor whether ban activity levels are also dynamically regulated in the PG, use was made of a ban sensor that expresses GFP under control of a ubiquitously active tubulin promoter and carries two perfect ban fixation sites in its 3' UTR thus making it repressed in the presence of the miRNA. A control sensor lacking the fixation sites showed high GFP expression both in early and late larval PGs. ban sensor levels, however, were low in the PG of second- and early third-instar larvae and considerably increased in wandering third-instar larvae. This observation leads to the proposal that high ban activity in young larvae contributes to the maintenance of low ecdysone titers and the promotion of systemic growth, whereas reduced activity in late PGs contributes to the generation of the ecdysone peak, the cessation of growth, and entry into metamorphosis (Boulan, 2013).

What is the upstream signal that regulates ban activity? The conserved insulin/insulin growth factor (IGF) signaling pathway directly promotes growth in target tissues and is the main relay to couple body growth to nutritional state. In young feeding larvae, insulin signaling in the PG also promotes the basal production of ecdysone, which in turn inhibits body growth. This buffering mechanism, based on the antagonistic action of insulin and ecdysone, modulates final body size in response to nutritional changes. Interestingly, ban activity levels were strongly reduced in early PGs expressing different transgenes that activate the insulin pathway. Increased levels of circulating Dilp2 also reduced ban activity in early PGs, as monitored by increased expression of the ban sensor. Thus, insulin signaling represses ban activity in ecdysone-producing cells (Boulan, 2013).

Thanks to a nutrient-sensing mechanism in the fat body, the equivalent to the vertebrate liver, food conditions control the secretion or expression of brain-derived Dilps, which are the main systemic supply of this hormone during the growth period. Consistent with the repression caused by increased insulin signaling, young feeding larvae growing on amino acid-rich medium showed a clear decrease in ban activity in PG cells. This reduction depended on the enhanced activity of Dilps, because the inhibition of insulin signaling in the PG was sufficient to restore ban activity to normal levels (Boulan, 2013).

In order to address whether ban mediates the action of insulin in regulating ecdysone production, genetic interactions were performed in gain- and loss-of-function conditions. Remarkably, the reduced body size phenotype obtained by enhanced insulin signaling in the PG via several transgenes was completely rescued by simultaneously increasing ban levels in these cells. Given the fact that this rescue implies a much greater effect on body size than the overexpression of UAS-ban transgene alone, it was concluded that the effects of ban and insulin signaling are not additive but rather epistatic. This conclusion is further supported by the observation that modulation of insulin signaling in the PG no longer affected body size in a banΔ1 mutant background. Altogether, these results indicate that the regulation of ecdysone production by insulin signaling relies on the modulation of ban activity in PG cells (Boulan, 2013).

So far, these results unravel a novel role of ban in promoting larval body growth by reducing ecdysone production. In contrast, ban was initially identified by its capacity to induce organ growth in a cell-autonomous manner. That finding prompted an exploration of the contribution of the systemic and cell-autonomous activities of ban to organ growth. The imaginal discs of Drosophila are epithelial sacs that grow in feeding larvae to give rise after metamorphosis to the ectodermal structures of the adult flies, such as legs, wings, or eyes. In banΔ1 mutant larvae, the size of the wing imaginal discs was strongly reduced when compared to control animals. Targeted expression of ban in the PG partially rescued the wing growth defects observed in ban mutant larvae. This result supports the proposal that both the systemic and cell-autonomous activities of ban are required to promote organ growth (Boulan, 2013).

In conclusion, these results establish that ban promotes systemic growth by inhibiting the synthesis of the steroid hormone ecdysone. During the growth period, ban mediates the insulin-dependent regulation of ecdysone production and therefore acts as a buffering mechanism to adjust final body size in response to nutrient availability. Such a crosstalk between insulin and steroid hormones and its impact on the modulation of growth and developmental decisions are also observed in Caenorhabditis elegans. Depending on environmental conditions, the juvenile form of C. elegans either enters maturation to give rise to an adult worm or arrests development to form a dauer larva, a state that is specifically adapted for survival. This decision is determined by the levels of the steroid hormone dafachronic acid (DA). Mutations that enhance insulin signaling, thereby mimicking a favorable environment, increase DA levels and cause animals to become incapable of forming dauer larvae. Remarkably, the deletion of ban orthologs in C. elegans (the mir-58 family) causes severe growth defects and prevents entry into the dauer state under environmental stress (Alvarez-Saavedra, 2010). On the basis of these observations, it is proposed that the role of ban in preventing the production of steroid hormones in function of insulin and nutrient levels might be conserved in other organisms in order to regulate body growth and maturation (Boulan, 2013).

Local requirement of the Drosophila insulin binding protein Imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. This study shows that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2 - FlyBase name: Ecdysone-inducible gene L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together these results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions (Sarraf-Zadeh, 2013).

In higher organisms, the duration of the juvenile stage needs to be variable to ensure the development of a healthy and fertile adult. Environmental stresses, such as adverse nutritional conditions, can delay development until a critical weight is reached. Additional checkpoints ensure that increased growth rates, induced by ideal nutritional conditions, do not lead to a premature passage to the adult stage. In Drosophila, the juvenile growth stage is terminated by pupae formation at the end of the third larval instar. Larval/pupal transition is induced by a pulse of the steroid hormone ecdysone produced by the PG (Sarraf-Zadeh, 2013).

Genetic manipulations of the Drosophila PG revealed the requirements of the IIS, Target of Rapamycin (TOR) and PTTH pathways to control ecdysone production . Recently, IIS dependent growth of the PG has been identified as an additional factor controlling ecdysone production. Overexpression of PI3K, a positive regulator of IIS, leads to premature, increased ecdysone production resulting in a shortened L3 stage and early pupariation. By contrast, overexpression of negative regulators of IIS in the PG delays pupariation caused by lowered and delayed ecdysone production. Reduction of whole organism IIS activity does not change critical weight but delays its attainment. In contrast, ablation of PTTH neurons induces a severe shift in critical weight, suggesting that these neurons play an important role in setting this parameter. When larvae reach the critical weight, PTTH is released on the PG and induces transcription of genes involved in ecdysone production. However, PTTH expression is not modified upon nutritional restriction, indicating that PTTH signaling does not mediate starvation induced developmental delay. Signaling via TOR, the downstream kinase of IIS, links nutritional information to ecdysone production, since starvation induced developmental delay can partially be rescued by upregulating TOR activity in the PG. This suggests that downregulating TOR signaling upon starvation desensitizes the PG for PTTH signals, resulting in delayed ecdysone production. The present study shows that increased IIS activity in the PG due to Imp L2 LOF rescues the delay caused by malnutrition to a large extent, indicating that low IIS also renders the PG irresponsive to the PTTH signal. Whether the effects of low IIS in the PG are mediated by TOR or whether the two pathways act independently remains to be elucidated (Sarraf-Zadeh, 2013).

Evidence is presented for a number of Imp L2 expressing neurons to act as possible regulators of IIS activity in the PG. High Imp L2 levels in the hemolymph can be excluded as possible inhibitors of IIS signaling in the PG, since increasing hemolymph levels of Imp L2 failed to reduce size and IIS activity of PG cells, but resulted in a strong size decrease of the whole organism. On the other hand, increasing Imp L2 levels in Imp L2 positive neurons targeting the PG causes a massive decrease in PG size and lowers IIS activity within PG cells. These results support the idea that the PG does not receive information about the nutritional state of the organism through the hemolymph but rather from Imp L2 expressing neurons. Thus, this work reveals a novel local function of the negative growth regulator Imp L2 in controlling IIS activity and ecdysone production in the PG. This finding reveals a novel mechanism for the spatial regulation of IIS: through locally restricted effects of Imp L2, diverse tissues can be effectively subjected to different levels of IIS (Sarraf-Zadeh, 2013).

Interestingly, the ability of IIS to coordinate growth with development seems to be conserved throughout evolution. In humans, the onset of puberty is linked to the nutritional state, leading to early puberty in well fed western societies. In contrast, juvenile females suffering from type I diabetes mellitus display a notable delay in menarche, indicating that decreased IIS also delays maturation in humans. Moreover, in Caenorhabditis elegans, malnutrition during the first larval stage leads to developmental arrest by inducing dauer formation, which is a larval stage best adapted for survival under adverse environmental conditions. Mutations reducing IIS pathway activity lead to dauer formation independent of the nutritional state. Hence, different phyla developed similar strategies to cope with adverse nutritional conditions during the juvenile state. When IIS activity is below a certain threshold, development is attenuated until sufficient nutrients are available, to ensure the formation of healthy and fertile adults. In Drosophila larval malnutrition leads to delayed pupariation, due to decreased IIS activity in the PG which in turn delays the production of the steroid hormone ecdysone (Sarraf-Zadeh, 2013).

Steroid hormones also play an important role in human development. In cases of human hypogonadism, puberty is prolonged, which can lead to abnormally tall adults if not treated with steroid substitutes. Referring the current data to the human system, the putative Imp L2 homolog IGFBP 7 (also known as IGFBP rP1) also displays a very diverse protein expression pattern, indicating a specialized function in different organs. Amongst other tissues, IGFBP 7 is expressed in different regions of the human brain, leading to the speculation that it might act as a local regulator of steroid production as well (Sarraf-Zadeh, 2013).

In summary, the data provides novel insights into the coupling of developmental cues to nutritional state. Since IIS and steroid hormones play evolutionarily conserved roles in regulating growth and development, the findings on the local function of the insulin binding protein Imp L2 in controlling ecdysone production might be of general interest (Sarraf-Zadeh, 2013).

Eclosion gates progression of the adult ecdysis sequence of Drosophila

Animal behavior is often organized into stereotyped sequences that promote the goals of reproduction, development, and survival. However, for most behaviors, the neural mechanisms that govern the order of execution of the motor programs within a sequence are poorly understood. An important model in understanding the hormonal determinants of behavioral sequencing is the ecdysis sequence, which is performed by insects at each developmental transition, or molt. The adult ecdysis sequence in Drosophila includes the emergence of the insect from the pupal case followed by expansion and hardening of the wings. Wing expansion is governed by the hormone bursicon, and stimulation of the bursicon-expressing neurons in newly eclosed flies induces rapid wing expansion. This study shows that that such stimulation delivered prior to eclosion has no immediate effect, but does cause rapid wing expansion after eclosion if the stimulus is delivered within 40 min of that event. A similar delayed effect was observed upon stimulation of a single pair of bursicon-expressing neurons previously identified as command neurons for wing expansion. It is concludes that command neuron stimulation enables the motor output pathway for wing expansion, but that this pathway is blocked prior to eclosion. By manipulating the time of eclosion, this study demonstrates that some physiological process tightly coupled to adult ecdysis releases the block on wing expansion. Eclosion thus serves as a behavioral checkpoint and complements hormonal mechanisms to ensure that wing expansion strictly follows eclosion in the ecdysis sequence (Peabody 2013).

Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster

Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, a screen was conducted of 545 splice-trap lines and 28 loci were identified that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and SHEP proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple pre-synaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, though not entirely, normal locomotor behavior. Thus, shep plays an important role in the metamorphic development of many neurons (Chen, 2014).

Peptidergic neurons produce small peptides, called neuropeptides, which are secreted within the nervous system to influence the activity of other neurons or into the blood to act on other tissues. Through these targets, neuropeptides regulate a wide range of processes, which include development, feeding, growth, aggression, reproduction and learning and memory. One of the first genes identified to play a specific role in the development of peptidergic neurons was dimmed (dimm), which encodes a basic helix-loop-helix transcription factor that is required for the differentiation of diverse peptidergic neurons. Dimm is a key regulator of expression of the neuropeptide biosynthetic enzyme, peptidylglycine-alphahydroxylating monooxygenase (Phantom or Phm), and it promotes the differentiation of neurosecretory properties in many neurons. Both Dimm and Phm are expressed widely and specifically in peptidergic neurons. In fact, Dimm was first identified by virtue of its pattern of peptidergic neuron expression through an enhancer-trap screen. Similar expression pattern-based strategies may be useful for identification of other factors critical for peptidergic neuron development (Chen, 2014).

This study sought to identify similar factors through a splice-trap screen for genes with peptidergic cell-specific expression patterns. 28 insertions were identified with different patterns of peptidergic cell reporter gene expression, driven by P element splicetrap insertions in specific loci. These insertions drove reporter expression in insulin-like peptide 2 (ILP2), crustacean cardioactive peptide (CCAP)/bursicon, -RFamide, Furin 1, and leucokinin (LK) cells and often caused defects typical of disrupted neuropeptide signaling. Thus, all 28 of these genes are strong candidate regulators of peptidergic cell development or function (Chen, 2014).

One of the splice-trap insertions was mapped to an exon of the alan shepard (shep) gene, and this insertion was chosen for further analysis because it displayed an expression pattern that was highly similar to Phm and Dimm. shep in situ hybridization and anti-Shep immunostaining later revealed that both the shep mRNA and Shep protein expression is enriched in most neurons, yet shep mutants displayed defects in adult eclosion and wing expansion that suggested specific disruptions in signaling by bursicon and other neuropeptides. Consistent with these behavioral phenotypes, the shep mutant bursicon neurons had smaller somata, fewer axon branches, and smaller and fewer neuroendocrine boutons, and all of these phenotypes were rescued by expression of a wild-type shep cDNA. Interestingly, pan-neuronal RNA interference to shep produced smaller CNS neuropils and defects in general locomotor behaviors, such as flipping and climbing. Most of the locomotor phenotypes were restricted to the adult stage, and the effects of shep mutations on neuronal growth were restricted to pupal development. Thus, shep regulates metamorphic growth of the bursicon neurons, and it may also serve as a general regulator of neuronal growth during metamorphic remodeling (Chen, 2014).

Anti-Shep immunostaining and additional shep reporter genes confirmed expression in peptidergic neurons, but these markers and shep in situ hybridization also revealed widespread expression in the CNS, with much lower expression in other tissues. Shep is orthologous to the c-myc single-strand binding protein, MSSP-2: Previous studies have described shep as homologous to the vertebrate genes, Rbms2/Scr3 (Armstrong, 2006) or Rbms1/Scr2/MSSP-2. Phylogenetic analysis supports the placement of Shep in the MSSP family, with the ELAV family of RNA-binding proteins being the next most closely related. In general, MSSP proteins contain RNA recognition motifs and have been found in vertebrates to bind DNA, RNA, or proteins to regulate a variety of biological processes, including DNA polymerization, gene expression, cell transformation, and apoptosis. In Drosophila, Shep interacts with the insulator proteins Mod(mdg4)2.2 and Su(Hw) to negatively regulate chromosomal insulator activities, specifically in the CNS (Matzat, 2012). These molecular insights suggest a gene regulatory mechanism by which Shep may control aspects of the metamorphic development of the bursicon neurons, as well as other neurons that contribute to the overall structure of adult brain neuropils (Chen, 2014).

The shep mutant defects in wing expansion presented an opportunity to define cellular functions of Shep in an experimentally accessible cell type, the bursicon neurons. In shep mutants, a reduction was observed in the post-pruning growth of the bursicon neurons during metamorphosis, resulting in smaller somata and less branching in the peripheral axon arbor in pharate adult animals. Interestingly, the regulation of bursicon neuron growth by shep was stage-dependent. Defects were observed in bursicon neuron soma growth and axon branching during metamorphosis in hypomorphic shep mutant animals of multiple genotypes, including shepBG00836/shepBG00836, shepExel6103/shepExel6104 and shepBG00836/shepED210. However, in each of these genotypes, the larval cellular morphologies were normal. Other behavioral defects were observed that suggested that the metamorphosis-specific actions of Shep were not limited to the bursicon neurons. For example, the most severe shep loss-of-function genotype tested was elav>shep-RNAi, Dicer-2, but elav>shep-RNAi, Dicer-2 larvae displayed normal crawling distances and self-righting behaviors, while this genotype showed lethality in the late pupal stages and severe locomotor defects in adult animals. Associated with this increase during metamorphosis in the dependence of the nervous system on shep activity, there is also a marked increase in the levels of shep expression at the onset of metamorphosis (Chen, 2014).

These results provide indirect evidence to suggest that an increase in shep expression during the pupal stage may support neuronal remodeling or other aspects of neuronal function and development in diverse neurons during metamorphosis. Although most of the larval behaviors assayed were unaffected in shep mutant animals, one behavioral phenotype in was observed in elav>shep-RNAi, Dicer-2 larvae, namely a tendency to remain in the center of the apple juice-agarose plate while making many sharp turns along the path of locomotion. Based on anti-Shep immunostaining, UAS-shep-RNAi, Dicer-2 provided a more complete knockdown of anti-Shep immunostaining in the CNS than shep RNAi without UAS-Dicer-2 or in shepBG00836 homozygotes or shepBG00836/shepED210 mutant larvae. Moreover, shep RNAi without UAS-Dicer-2 led to a greater knock-down of Shep in Western blots than shepExel6103/shepExel6104 (Matzat, 2012). Taken together with the above observation that many of the weaker shep loss-of-function genotypes had defects that were only manifest in adults, these findings suggest that shep plays a stage-dependent (largely metamorphosis-specific) role in the maintenance, function, or development of the nervous system (Chen, 2014).

The Shep expression pattern and shep mutant phenotypes reported in this study are consistent with broad actions of this protein in neuronal development and functions throughout the nervous system. Pan-neuronal loss of shep resulted in late-pupal lethality and reduced adult life span under both fed and starved conditions, as well as diverse developmental and behavioral defects, including failure to complete wing expansion, uncoordinated and weakened adult locomotion, reduced neuropil areas, and altered mating behaviors. Other groups have also shown defects in gravitaxis and reduced starvation resistance in shep mutants (Armstrong, 2006; Harbison, 2004; Chen, 2014 and references therein).

Such widespread actions may also explain the partial rescue of the mating defects by UAS-shep expression in shepBG00836/shepED210 females. Although the possibility cannot be excluded that other Shep isoforms in addition to Shep-E/G (used to create UAS-shep) str necessary to support the normal function of the post-copulatory grooming circuits, it is also possible that neurons required for female receptivity to the male may have been included in the shepBG00836 expression pattern used to drive shep rescue, whereas the neurons involved in normal post-copulatory grooming behaviors are not (Chen, 2014).

The observation of several seemingly independent behavioral defects (e.g., gravitaxis and female receptivity to mating) and reduced neuropil areas, taken together with the cellular defects described in shep-mutant bursicon neurons, suggests that Shep may have pleiotropic effects on neurite development or other processes throughout the CNS. Such pleiotropic effects of shep mutations in the CNS may be due to the loss of Shep suppression of widely distributed chromatin insulator complexes (Matzat, 2012), so as to establish altered chromatin states and gene expression, potentially in multiple signaling pathways controlling a range of developmental and physiological events. In addition, some of the adult shep loss-of-function phenotypes, such as reduced lifespan and altered mating behaviors, may reflect adult-specific (acute) effects of Shep on neuronal activity. Alternatively, the metamorphosis-specific regulation of neurite branching and cell growth in the bursicon neurons may be representative of the actions of Shep in many neuronal cell types. It will be important in future studies to distinguish among these models, as the results demonstrate that Shep is a general regulator of the postembryonic development of mature neurons (Chen, 2014).

CDK8-Cyclin C mediates nutritional regulation of developmental transitions through the Ecdysone receptor in Drosophila

EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition (Xie, 2015).

In animals, the amount of juvenile growth is controlled by the coordinated timing of maturation and growth rate, which are strongly influenced by the environmental factors such as nutrient availability. This is particularly evident in arthropods, such as insects, arachnids and crustaceans, which account for over 80% of all described animal species on earth. Characterized by their jointed limbs and exoskeletons, juvenile arthropods have to replace their rigid cuticles periodically by molting. In insects, the larval-larval and larval-pupal transitions are controlled by the interplay between juvenile hormone (JH) and steroid hormone ecdysone. Drosophila has been a powerful system for deciphering the conserved mechanisms that regulate hormone signaling, sugar and lipid homeostasis, and the molecular mechanisms underlying the nutritional regulation of development. In Drosophila, all growth occurs during the larval stage when larvae constantly feed, and as a result their body mass increases approximately 200-fold within 4 d, largely due to de novo lipogenesis. At the end of the third instar, pulses of ecdysone, combined with a low level of JH, trigger the larval-pupal transition and metamorphosis. During this transition, feeding is inhibited, and after pupariation, feeding is impossible, thus the larval-pupal transition marks when energy metabolism is switched from energy storage by lipogenesis in larvae to energy utilization by lipolysis in pupae (Xie, 2015).

The molecular mechanisms of ecdysone-regulated metamorphosis and developmental timing have been studied extensively in Drosophila. Ecdysone binds to the Ecdysone Receptor (EcR), which heterodimerizes with Ultraspiracle (USP), an ortholog of the vertebrate Retinoid X Receptor (RXR). By activating the expression of genes whose products are required for metamorphosis, ecdysone and EcR-USP are essential for the reorganization of flies' body plans before emerging from pupal cases as adults. Despite the tremendous progress in understanding of the physiological and developmental effects of EcR-USP signaling, the molecular mechanism of how the EcR-USP transcription factor interacts with the general transcription machinery of RNA polymerase II (Pol II) and stimulates its target gene expression remains mysterious. EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithorax-related (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression, it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression. In addition, it remains poorly understood how EcR activates transcription correctly after integrating nutritional and developmental cues (Xie, 2015).

The multisubunit Mediator complex serves as a molecular bridge between transcriptional factors and the core transcriptional machinery, and is thought to regulate most (if not all) of Pol II-dependent transcription. Biochemical analyses have identified two major forms of the Mediator complexes: the large and the small Mediator complexes. In addition to a separable 'CDK8 submodule', the large Mediator complex contains all but one (MED26) of the subunits of the small Mediator complex. The CDK8 submodule is composed of MED12, MED13, CDK8, and CycC. CDK8 is the only enzymatic subunit of the Mediator complex, and CDK8 can both activate and repress transcription depending on the transcription factors with which it interacts. Amplification and mutation of genes encoding CDK8, CycC, and other subunits of Mediator complex have been identified in a variety of human cancers, however, the function and regulation of CDK8-CycC in non-disease conditions remain poorly understood. CDK8 and CycC are highly conserved in eukaryotes, thus analysis of the functional regulation of CDK8-CycC in Drosophila is a viable approach to understand their activities (Xie, 2015).

Previous, work has shown that CDK8-CycC negatively regulates the stability of sterol regulatory element-binding proteins (SREBPs) by directly phosphorylating a conserved threonine residue. This study now reports that CDK8-CycC also regulates developmental timing in Drosophila by linking nutrient intake with EcR-activated gene expression. Homozygous cdk8 or cycC mutants resemble EcR mutants in both pupal morphology and retarded developmental transitions. Despite the elevation of both EcR and USP proteins in cdk8 or cycC mutants, genome-wide gene expression profiling analyses reveal systematic down-regulation of EcR-target genes, suggesting the CDK8-CycC defect lies between the receptor complex and transcriptional activation. CDK8-CycC is required for EcR-USP transcription factor binding to EcR target genes. Mass spectrometry analysis for proteins that co-immunoprecipitate with EcR and USP has identified multiple Mediator subunits, including CDK8 and CycC, and yeast two-hybrid assays have revealed that CDK8 and Med14 can directly interact with the EcR-AF1 domain. Furthermore, the dynamic changes of CDK8, EcR, USP, and SREBP correlated with the fundamental roles of SREBP in regulating lipogenesis and EcR-USP in regulating metamorphosis during the larval–pupal transition. Importantly, it was shown that starving the early third instar larvae causes precocious increase of CDK8, EcR and USP proteins, as well as premature inactivation of SREBP; whereas refeeding of the starved larvae reduces CDK8, EcR, and USP proteins, but potently stimulates SREBP activity. These results suggest a dual role of CDK8-CycC, linking nutrient intake to de novo lipogenesis (by inhibiting SREBP) and developmental signaling (by regulating EcR-dependent transcription) during the larval–pupal transition (Xie, 2015).

Through EcR-USP, ecdysone plays pivotal roles in controlling developmental timing in Drosophila. This study shows that cdk8 or cycC mutants resemble EcR-B1 mutants and CDK8-CycC is required for proper activation of EcR-target genes. Molecular and biochemical analyses suggest that CDK8-CycC and the Mediator complexes are directly involved in EcR-dependent gene activation. In addition, the protein levels of CDK8 and CycC are up-regulated at the onset of the wandering stage, closely correlated with the activation of EcR-USP and down-regulation of SREBP-dependent lipogenesis during the larval–pupal transition. Remarkably, starvation of the feeding larvae leads to premature up-regulation of CDK8 and EcR-USP, and precocious down-regulation of SREBP, while refeeding of the starved larvae results in opposite effects on the CDK8-SREBP/EcR network. Thus, it is proposed that CDK8-CycC serves as a key mediator linking food consumption and nutrient intake to EcR-dependent developmental timing and SREBP-dependent lipogenesis during the larval–pupal transition (Xie, 2015).

The Mediator complex is composed of up to 30 different subunits, and biochemical analyses of the Mediator have identified the small Mediator complex and the large Mediator complex, with the CDK8 submodule being the major difference between the two complexes. Several reports link EcR and certain subunits of the Mediator complex. For example, Med12 and Med24 were shown to be required for ecdysone-triggered apoptosis in Drosophila salivary glands. It was recently reported that ecdysone and multiple Mediator subunits could regulate cell-cycle exit in neuronal stem cells by changing energy metabolism in Drosophila, and specifically, EcR was shown to co-immunoprecipitate with Med27. However, exactly how Mediator complexes are involved in regulating EcR-dependent transcription remains unknown. The current data suggest that CDK8 and CycC are required for EcR-activated gene expression. Loss of either CDK8 or CycC reduced USP binding to EcR target promoters, diminished EcR target gene expression, and delayed developmental transition, which are reminiscent of EcR-B1 mutants. Importantly, mass spectrometry analysis for proteins that co-immunoprecipitate with EcR or USP has identified multiple Mediator subunits, including all four subunits of the CDK8 submodule (Xie, 2015).

Taken together, previous works and the present work highlight a critical role of the Mediator complexes including CDK8-CycC in regulating EcR-dependent transcription. How does CDK8-CycC regulate EcR-activated gene expression? Biochemical analyses show that CDK8 can interact with EcR and USP in vivo and that CDK8 can directly interact with EcR-AF1. These observations, together with the current understanding of how nuclear receptors and Mediator coordinately regulate transcription, suggest that CDK8-CycC may positively and directly regulate EcR-dependent transcription. Yeast two-hybrid analysis indicates that the recruitment of CDK8-CycC to EcR-USP can occur via interactions between CDK8 and the AF1 domain of EcR. Interestingly, this assay also revealed a direct interaction between EcR-AF1 and a fragment of Med14 that contains the LXXLL motif. In future work, it will be interesting to determine whether CDK8 and Med14 compete with each other in binding with the EcR-AF1, whether they interact with EcR-AF1 sequentially in activating EcR-dependent transcription, and how the Mediator complexes coordinate with other known EcR cofactors in regulating EcR-dependent gene expression (Xie, 2015).

In cdk8 or cycC mutants, the binding of USP to the promoters of the EcR target genes is significantly compromised, even though nuclear protein levels of both EcR and USP are increased. It is unclear how CDK8-CycC positively regulates EcR-USP binding to EcREs near promoters. CDK8 can directly phosphorylate a number of transcription factors, such as Notch intracellular domain, E2F1, SMADs, SREBP, STAT1, and p53. Interestingly, the endogenous EcR and USP are phosphorylated at multiple serine residues, and treatment with 20E enhances the phosphorylation of USP. Protein kinase C has also been proposed to phosphorylate USP. It will be interesting to determine whether CDK8 can also directly phosphorylate either EcR or USP, thereby potentiating expression of EcR target genes and integrating signals from multiple signaling pathways (Xie, 2015).

Although a direct role for CDK8-CycC to regulate EcR-USP activated gene expression is favored, it was not possible to exclude the potential contribution of impaired biosynthesis of 20E to the developmental defects in cdk8 or cycC mutants. For example, the expression of genes involved in synthesis of 20E, such as sad and spok, is significantly reduced in cdk8 or cycC mutant larvae. Indeed, the ecdysteroid titer is significantly lower in cdk8 mutants than control from the early L3 to the WPP stages, and feeding the cdk8 mutant larvae with 20E can partially reduce the retardation in pupariation. Nevertheless, impaired ecdysone biosynthesis alone cannot explain developmental defects that were characterized in this report for the following reasons. First, feeding cdk8 or cycC mutants with 20E cannot rescue the defects in pupal morphology, developmental delay, and the onset of pupariation. Second, the expression of EcRE-lacZ reporter in cdk8 or cycC mutant salivary glands cannot be as effectively stimulated by 20E treatment as in control. Third, knocking down of either cdk8 or cycC in PG did not lead to obvious defects in developmental timing. Therefore, the most likely scenario is that the cdk8 or cycC mutants are impaired not only in 20E biosynthesis in the PG, but also in EcR-activated gene expression in peripheral tissues. Defects in either ecdysone biosynthesis or EcR transcriptional activity will generate the same outcome: diminished expression of the EcR target genes, thereby delayed onset of pupariation (Xie, 2015).

How CDK8-CycC regulates biosynthesis of ecdysone in PG remains unknown. Several signaling pathways have been proposed to regulate ecdysone biosynthesis in Drosophila PG, including PTTH and Drosophila insulin-like peptides (dILPs)-activated receptor tyrosine kinase pathway and Activins/TGFβ signaling pathway. Interestingly, CDK8 has been reported to regulate the transcriptional activity of SMADs, transcription factors downstream of the TGFβ signaling pathway, in both Drosophila and mammalian cells. Thus, it is conceivable that the effect of cdk8 or cycC mutation on ecdysone biosynthesis may due to dysregulated TGFβ signaling in the PG (Xie, 2015).

An effort to explore the potential role of food consumption and nutrient intake on CDK8-CycC has resulted an unexpected observation that the protein level of CDK8 is strongly influenced by starvation and refeeding: starvation potently increased CDK8 level, while refeeding has opposite effect, and both occur post-transcriptionally. The importance of this observation is highlighted in two aspects. First, considering the generally repressive role of CDK8 on Pol II-dependent gene expression, up-regulation of CDK8 may provide an efficient way to quickly tune down most of the Pol II-dependent transcription in response to starvation; while down-regulation of CDK8 in response to refeeding may allow many genes to express when nutrients are abundant. Second, it will be necessary to test whether the effects of nutrient intake on CDK8-CycC is conserved in mammals. If so, considering that both CDK8 and CycC are dysregulated in a variety of human cancers, the effects of nutrient intake on CDK8 may have important implications in not only understanding of the effects of nutrients on tumorigenesis, but also providing nutritional guidance for patients with cancer (Xie, 2015).

Major dietary components including carbohydrates, lipids, and proteins, can strongly influence the developmental timing in Drosophila. Excessive dietary carbohydrates repress growth and potently retard the onset of pupariation. One elegant model proposed to explain how high sugar diet delays developmental timing is that high sugar diet reduces the activity of the Target of Rapamycin (TOR) in the PG, thereby reducing the secretion of ecdysone and delaying the developmental transition. Previously, it was reported that insulin signaling could down-regulate CDK8-CycC, and that ectopic expression of CycC could antagonize the effect of insulin stimulation in mammalian cells, as well as the effect of refeeding on the expression of dFAS in Drosophila (Zhao, 2012). Although the mRNA levels of TOR and insulin receptor (InR) are not significantly affected in cdk8 or cycC mutants, it is necessary to further study whether and how different dietary components may regulate CDK8-CycC in the future (Xie, 2015).

Developmental genetic analyses of the cdk8 and cycC mutants have revealed major defects in fat metabolism and developmental timing. De novo lipogenesis, which is stimulated by insulin signaling, contributes significantly to the rapid increase of body mass during the constant feeding larval stage. This process is terminated by pulses of ecdysone that trigger the wandering behavior at the end of the L3 stage, followed by the onset of the pupariation. Insulin and ecdysone signaling are known to antagonize each other, and together determine body size of Drosophila. The genetic interaction is established, but the detailed molecular mechanisms are not. The SREBP family of transcription factors controls the expression of lipogenic enzymes in metazoans and the expression of cholesterogenic enzymes in vertebrates. Previous work shows that CDK8 directly phosphorylates the nuclear SREBP proteins on a conserved threonine residue and promotes the degradation of nuclear SREBP proteins. Consistent with the lipogenic role of SREBP and the inhibitory role of insulin to CDK8-CycC, the transcriptional activity of SREBP is high while the levels of CDK8-CycC and EcR-USP are low prior to the onset of wandering stage. Subsequently during the wandering and non-mobile, non-feeding pupal stage, the transcriptional activity of SREBP is dramatically reduced, accompanied by the significant accumulation of CDK8-CycC and EcR-USP (Xie, 2015).

The causal relationship of these phenomena was further tested by starvation and refeeding experiments. On the one hand, it was observed that the levels of CDK8, EcR and USP are potently induced by starvation, while the mature SREBP level and the transcriptional activity of SREBP are reduced by starvation. Starvation of larvae prior to the two nutritional checkpoints in early L3, known as minimum viable weight and critical weight, which are reached almost simultaneously in Drosophila, will lead to larval lethality; while starvation after larvae reach the critical weight will lead to early onset of pupariation and formation of small pupae. Thus, this nutritional checkpoint ensures the larvae have accumulated sufficient growth before metamorphosis initiation. If the status with high CDK8, EcR, and USP is regarded as an older or later stage, these results indicate that starvation shifts the regulatory network precociously (see Model for the CDK8-SREBP/EcR regulatory network). On the other hand, the current analyses of refed larvae show that refeeding potently reduced the levels of CDK8, EcR and USP. If the status with low CDK8, EcR, and USP is considered as a younger or earlier stage, these results indicate that refeeding delays the activation of this network, which is consistent with the model and delayed pupariation as observed. Taken together, these results based on starved and refed larvae suggest that CDK8-CycC is a key regulatory node linking nutritional cues with de novo lipogenesis and developmental timing (Xie, 2015).

The larval-pupal transition is complex and dynamic. Although the expression of SREBP target genes fit well with the predicted effects of starvation and refeeding, the expression of EcR targets during the stage that was analyzed in this study does not reflect the changes in the protein levels of EcR and USP. It is reasonable to consider that CDK8-CycC and EcR-USP are necessary, but not sufficient, for the activation of EcR target genes. One possibility is that there is a delay on synthesis of 20E or other cofactors that are required for EcR-activated gene expression in response to starvation. Indeed, the 20E levels were measured during the first 16 hr of starvation, and no significant difference was observed between fed and starved larvae. It will be necessary to further analyze the effect of starvation on 20E synthesis at later time points in the future (Xie, 2015).

Taken together, a model is proposed whereby CDK8-CycC functions as a regulatory node that coordinates de novo lipogenesis during larval stage and EcR-dependent pupariation in response to nutritional cues. It is likely that pulses of 20E synthesized in the PG, and subsequent behavioral change from feeding to wandering, ultimately trigger the transition from SREBP-dependent lipogenesis to EcR-dependent pupariation. The opposite effects of CDK8-CycC on SREBP- and EcR-dependent gene expression suggest that the role of CDK8 on transcription is context-dependent (Xie, 2015).

In conclusion, this study illustrates how CDK8-CycC regulates EcR-USP-dependent gene expression, and the results suggest that CDK8-CycC may function as a regulatory node linking fat metabolism and developmental timing with nutritional cues during Drosophila development (Xie, 2015).

Smads and insect hemimetabolan metamorphosis

In contrast with Drosophila melanogaster, practically nothing is known about the involvement of the TGF-β signaling pathway in the metamorphosis of hemimetabolan insects. To partially fill this gap, the role of Smad factors in the metamorphosis of the German cockroach, Blattella germanica, was studied. In Drosophila, Mad is the canonical R-Smad of the BMP branch of the TGF-β signaling pathway, Smox is the canonical R-Smad of the TGF-β/Activin branch and Medea participates in both branches. In insects, metamorphosis is regulated by the MEKRE93 pathway, which starts with juvenile hormone (JH), whose signal is transduced by Methoprene-tolerant (Met), which stimulates the expression of Kruppel homolog 1 (Kr-h1) that acts to repress E93, the metamorphosis trigger. In B. germanica, metamorphosis is determined at the beginning of the sixth (final) nymphal instar (N6), when JH production ceases, the expression of Kr-h1 declines, and the transcription of E93 begins to increase. The RNAi of Mad, Smox and Medea in N6 of B. germanica reveals that the BMP branch of the TGF-beta signaling pathway regulates adult ecdysis and wing extension, mainly through regulating the expression of Bursicon, whereas the TGF-beta/Activin branch contributes to increasing E93 and decreasing Kr-h1 at the beginning of N6, crucial for triggering adult morphogenesis, as well as to regulating the imaginal molt timing (Santos, 2016).

adult structures

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