InteractiveFly: GeneBrief

Bursicon: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon [Fraenkel, 1962; Cottrell, 1962), but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon (Honegger, 2002), the Drosophila gene CG13419 was identified as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted Bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-ß (TGF-ß) and glycoprotein hormones. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased Bursicon bioactivity. In situ hybridization and immunocytochemistry reveal that Bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons also lack Bursicon bioactivity. These results indicate that CG13419 encodes Bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to be assigned a defined function in invertebrates. Mutants show that the spectrum of Bursicon actions is broader than formerly demonstrated (Dewey, 2004).

bursicon transcripts are expressed in a set of the neurons that contain CCAP. Transgenic Drosophila bearing targeted ablations of CCAP neurons (CCAP KO flies) are defective in wing expansion and tanning and have low survival to adulthood (Park, 2003). In situ hybridization of the CNS of CCAP KO larvae using a bursicon antisense probe shows a significant reduction in the number of labeled neurons, as would be expected if CCAP neurons express bursicon. In some preparations, a few surviving neurons weakly express the CG13419 message in the posterior VNS, consistent with previous findings (Park, 2003). Similarly, it was predicted that homogenates of CNSs from the CCAP KO flies would not contain bursicon bioactivity if tested in the neck-ligated Sarcophaga bioassay. Indeed, homogenates from CCAP KO flies contain little or no bursicon activity in the Sarcophaga bioassay, whereas homogenates of the relevant control flies (LacZ) produced a score similar to that of wild-type flies (Dewey, 2004).

The phenotype of burs mutants is similar to that of rickets (rk) mutants, which also fail to sclerotize, pigment, and expand their wings (Baker, 2002). The rk gene encodes a G protein-coupled receptor with a large, leucine-rich extracellular domain and was proposed to be a Bursicon receptor. It belongs to a subfamily of receptors whose ligands include the vertebrate glycoprotein hormones (Baker, 2002), which are also cystine-knot proteins. The similarity in phenotype between burs and rk mutants suggests that they may represent the ligand and receptor for this hormonal signaling pathway. Nevertheless, flies that were transheterozygous for mutant alleles of both genes (rk⁴/+; burs^Z1091/+ and rk⁴/+; burs^Z5569/+) showed no defects in wing expansion or tanning. Additional detailed experiments will need to be carried out to determine whether these two molecules interact (Dewey, 2004).

The homology to peptide sequences from cockroach bursicon and other insect species, the phenotypes and reduction in Bursicon activity in mutants of this gene, and the localization of its transcripts to neurons known to produce bursicon in other insect species, make a convincing case that CG13419 encodes the tanning hormone Bursicon. Hence, 42 years after its discovery, the structure of the last of the classic insect developmental hormones has finally been elucidated. Bursicon is the first member of the cystine knot family of signaling molecules to be assigned a defined hormonal function in invertebrates. Knowledge of the molecular identity of Bursicon now provides tools for the analysis of its function, including the mechanism of its tightly timed action in triggering the biochemical events (Andersen, 1996) that lead to cuticle hardening. The colocalization of Bursicon with CCAP, a peptide that activates the ecdysis motor program, poses the fascinating problem of how these two hormones are differentially regulated to produce two temporally distinct behaviors. The understanding of the neuroendocrine control of cuticle sclerotization may also have potential for the development of novel insect-specific pest control agents and measures (Dewey, 2004).

Bursicon signaling mutations separate the epithelial-mesenchymal transition from programmed cell death during Drosophila melanogaster wing maturation

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial-mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation (Natzle, 2008. Full text of article).

Completion of wing morphogenesis in Drosophila is an active process that is part of the suite of events initiated following eclosion of the fly from the pupal case. These events are known to be controlled by release of Bursicon from specific neurons in the abdominal ganglion. This study investigated the possibility that the Bursicon/Rickets signaling pathway also plays a role in the events that remove the cellular epidermis from within the expanding wing. The approach differed from that of other labs that have studied programmed cell death in the wing in that a marker was simultaneously followed for nuclear breakdown associated with programmed cell death (DAPI chromatin staining) and another marker was followed for the behavior and integrity of adherens junctions between cells (Arm-GFP). Mutations in this signaling pathway separate the dissociation of the epithelial cells from initiation of programmed cell death. In wild-type wings, within about an hour after eclosion, redistribution of Arm-GFP from its location in the subapical adherens junctions to the cytoplasm is accompanied by changes in cell shape. Within the same time frame, chromatin condensation was also observed, indicating nuclear changes accompanying cell death, beginning in some nuclei and spreading to others. Loss-of-function mutations in several different components in the signaling pathway (rickets, bursicon, pupal) and a potential upstream component affecting the secretion of Bursicon (batone) all produce a similar phenotype marked by aberrant persistence of the epithelia with intact adherens junctions, extending at least to 24 hr post-eclosion. In contrast, in all of these mutant backgrounds, normal initiation of programmed cell death in the epithelial cells is observed with a time course similar to wild type. These results provide evidence for rk-dependent regulation of the EMT and existence of a cell-death-signaling pathway that is independent of signaling through the rk receptor. These data might also be consistent with a model in which rk activation is capable of initiating both the EMT and cell death, but cell death induction can also occur via an independent and redundant pathway in the absence of wild-type rk function (Natzle, 2008).

Elimination of the epithelial cells from the wing involves coordinated changes in the epithelia that involve delamination from the cuticle and loss of contacts between cells (Kiger, 2007), accompanied by rapid and widespread initiation of programmed cell death. Completion of the EMT is followed by removal of the cells from the wing. When the caspase inhibitor p35 is expressed in the epithelial cells, the cells complete the EMT and assume a rounded shape. A significant proportion of the disorganized cell population does not leave the wing. A recent study (Link, 2007) also demonstrated that mutations that block programmed cell death in the wing epithelium produce persistence of nuclear staining long past the time when cells are normally cleared from the wing. While Link did not employ methods that allowed visualization of the shape of whole cells or of adherens junctions, it is likely that persistence of the nuclei marked persistence of intact epithelial cells. These observations imply that completion of one or more steps in the cell death program are required in addition to the EMT for exit of epithelial cells from the wing. In this view, both activation of programmed cell death and the EMT are necessary for removal of the wing epithelium, but neither is sufficient. Link has proposed that the cells disintegrate within the wing and their debris is flushed from the wing by flowing hemolymph. The results presented in this study clearly show that EMT must accompany cell death for dispersal of the wing epithelium to occur. Programmed cell death without EMT does not cause loss of the intact wing epithelium (Natzle, 2008).

On the basis of the spindle shape assumed by individual epithelial cells and the appearance of cell streaming in wings dissected from the body, it has been suggested that active migration could facilitate removal of the cells from the wing, with the caveat that frequent grooming of the wings by the hind legs of the fly may be an important part of the process (Kiger, 2007). It is suggested that loss of adhesion and acquisition of motility, grooming, and hemolymph flow may all contribute in vivo to rapid removal of cells. It seems likely that cells in which p35 is expressed, or in which other functions necessary for programmed cell death have been impaired, may make or retain attachments to the cuticle or extracellular matrix that prevent their removal from the wing. The ability to modulate such attachments, which may be necessary for clearing cells from the wing, could depend on appropriate regulation of caspase activity. The Drosophila caspase Dronc regulates border cell migration in the ovary independently of its role in apoptosis. In the mouse, Caspase-8 is required for activation of calpain proteases, Rac, and lamellipodial assembly. A similar requirement for caspase activity could link progress of a cell death program to the release of wing epithelial cells from their substrate attachments following EMT (Natzle, 2008).

The evidence for independent activation of epithelial cell death and the EMT raises the question of how these events are coordinated at eclosion. A complete answer to this question will have to await identification of other signal(s) that initiate programmed cell death or modulate the EMT. It is likely that overall synchronization is controlled via activation of the peptidergic networks within the central nervous system and coordinated release of signals that regulate specific aspects of ecdysis behavior. For example, the same neurons that express Bursicon at eclosion also express other active signaling neuropeptides such as the crustacean cardioactive peptide (CCAP) and the myoinhibitory peptide (MIP) with potentially independent direct or indirect molecular targets. Sequential release of different regulators can also modulate a process, e.g., control of cuticle tanning via pre-eclosion CCAP stimulation of tyrosine hydroxylase (TH) protein accumulation followed by Bursicon-dependent activation of TH via phosphorylation after eclosion (Natzle, 2008).

Coordination of the post-eclosion behavior of cells within the wing epithelium also potentially involves signals to the wing epithelial cells, prior to their death and dispersal, directing additional functions such as construction of the matrix that bonds the two cuticular surfaces of the mature wing. For example, the Timp gene (tissue inhibitor of metalloproteinases) is expressed in wings of newly eclosed flies and is required for normal wing bonding. Homozygous Timp^-/Timp^- flies expand their wings and eliminate the epithelial cells normally; however, the cuticular surfaces of the wing do not bond. Small Timp^-/Timp^- clones in the epithelia, produced by mitotic recombination, exhibit no bonding defects, consistent with secretion and diffusion of Timp from neighboring Timp⁺/Timp^-cells (KIGER, 2007). In contrast, large Timp^-/Timp^- clones, produced by mitotic recombination using the Minute technique, generate improperly bonded wings similar to those of homozygous Timp^-/Timp^- flies. Thus, the genotype of the epithelial cells themselves affects bonding of the cuticular surfaces, establishing that the wing cells play a critical role in metabolizing and remodeling the wing extracellular matrix. This is in agreement with observations that premature death of the epithelial cells prevents bonding of the cuticular surfaces. The timing and initiation of these cellular behaviors that affect wing bonding may also be coordinated via signals generated by the ecdysis cascade (Natzle, 2008).

For survival of the fly following eclosion in the natural environment, it is important that the entire wing epidermis be removed quickly to ensure timely completion of wing blade bonding and formation of functional flight organs. It would be reasonable to postulate that imposition of an EMT along with initiation of programmed cell death would ensure a more rapid and uniform dispersal of the epidermis from the appendage. Indeed, it would appear that the genes for producing EMT form a cassette that can be deployed as needed during the evolution of developmental pathways: first during gastrulation, then during peripheral nervous system formation, and later in winged insects during wing maturation (Natzle, 2008).

Neuromodulatory connectivity defines the structure of a behavioral neural network

Neural networks are typically defined by their synaptic connectivity, yet synaptic wiring diagrams often provide limited insight into network function. This is due partly to the importance of non-synaptic communication by neuromodulators, which can dynamically reconfigure circuit activity to alter its output. This study systematically mapped the patterns of neuromodulatory connectivity in a network that governs a developmentally critical behavioral sequence in Drosophila. This sequence, which mediates pupal ecdysis, is governed by the serial release of several key factors, which act both somatically as hormones and within the brain as neuromodulators. By identifying and characterizing the functions of the neuronal targets of these factors, this study found that they define hierarchically organized layers of the network controlling the pupal ecdysis sequence: a modular input layer, an intermediate central pattern generating layer, and a motor output layer. Mapping neuromodulatory connections in this system thus defines the functional architecture of the network (Diao, 2017).

Using the Trojan exon method to selectively target populations of hormone receptor-expressing neurons for manipulation and monitoring of activity, this study investigated the neuromodulatory connectivity of the circuitry governing pupal ecdysis behavior in Drosophila. The sites of action of the neuromodulators ETH, Bursicon, and CCAP identify essential functional components of the network architecture, defining three hierarchically organized layers from the sites of hormonal initiation to the sites of motor neuron output. In addition, it was found that descending neuromodulatory signaling from the ETHR-expressing input layer not only governs the basic motor rhythms of the ecdysis sequence by modulating the intermediate CPG layer, but also modulates activity of the CCAP-R-expressing motor neurons of the output layerd. Neuromodulators thus act broadly within, as well as across, network layers. The finding that the functional architecture of the ecdysis network can be decoded from its patterns of neuromodulatory connectivity provides further evidence that characterizing neuromodulatory connectomes is a valuable strategy in elucidating neural networks (Diao, 2017).

Major components of the pupal ecdysis circuitry are shared by the three motor programs A schematic (ETHRB-expressing and non-CCAP/ETHRA neurons regulate Phase I) broadly augments existing models of the pupal ecdysis network, both by providing a more comprehensive description of the input layer than has previously been possible and by identifying the motor circuits on which this layer acts. A principal finding reported in this study is that the downstream targets of Bursicon and CCAP are shared components of the pupal ecdysis network and are used to generate all three motor rhythms. The results draw particular attention to the centrality of neurons that express the Bursicon receptor (Rk), which are absolutely required for all pupal ecdysis behavior. A role in central pattern generation is indicated both from the effects of their suppression, which eliminates all motor activity, and from their pattern of ETH1-induced Ca++ activity, which matches the phases of ecdysis behavior. The fact that ETH1-induced Ca++ activity is observed in the excised nervous system and thus in the absence of sensory feedback, demonstrates that it is centrally generated and further supports the identification of the VNC-Rk neurons as central pattern generators. Conclusive evidence that some or all VNC-Rk neurons participate in central pattern generation will require more precise observations and perturbations than those performed in this study, as will determining the functional roles of individual neurons. However, the preliminary observation that regions containing at most small numbers of VNC-Rk neurons exhibit activity that is phasically coupled to two or more motor patterns argues that the ecdysis circuitry includes multifunctional CPG neurons that express Rk and are subject to modulation by distinct input layer modules. Similar architectures have been described in other motor networks where two CPGs formed from overlapping pools of neurons can switch between activity states to generate distinct behaviors (Diao, 2017).

How input layer neurons modulate the pupal ecdysis CPG is exemplified by the control of Phase II by ETHRA/CCAP neurons. Direct activation of these neurons induces Phase II-like rhythmic activity in the VNC-Rk neurons, an observation that is easily explained if Bursicon secreted from ETHRA/CCAP neurons shifts the mode of activity of the VNC-Rk CPG. This mechanism is consistent with the neuromodulatory control of CPGs described in numerous other systems and accounts for the long-standing observation that CCAP- and Bursicon-expressing neurons are important for pupal ecdysis, including Phase II ('ecdysis') initiation and Phase I ('pre-ecdysis') termination. The CCAP- and Bursicon-expressing neurons are known to express additional neuropeptides, including Myoinhibitory Peptides and Allatostatin C, and it is likely that these neuromodulators also play a role in regulating these phases. The mixed activity patterns that define the transition from Phase II to Phase III ('post-ecdysis') are also readily interpreted as a period of bistability in which CPG modes transiently alternate, perhaps as Bursicon and/or other co-released neuromodulator concentrations fall (Diao, 2017).

In addition to neurons that switch CPG activity from Phase I to Phase II, the input layer must also contain neurons that initiate pupal ecdysis by inducing Phase I. The search for such neurons has focused primarily on those that express ETHRA, but no components of this group have yet been identified that are required for ecdysis initiation. To identify the ETH targets responsible for Phase I, ETHR-expressing neurons were systematically parsed into three, nearly mutually exclusive subsets that together cover the entire input layer. The results indicate that the largely uncharacterized neurons that express the B-isoform of ETHR are required to initiate Phase I, and that the non-CCAP/ETHRA neurons are important for maintaining that phase (Diao, 2017).

The essential role of ETHRB-expressing neurons in Phase I initiation is consistent with the significantly higher affinity for ETH peptides of ETHRB compared with ETHRA. ETHRB-expressing neurons may thus initiate Phase I by responding to rising titers of ETH earlier than neurons expressing ETHRA. How they regulate the VNC-Rk CPG neurons remains to be determined, but their mechanism of action appears to be different from that of the ETHRA/CCAP neurons insofar as the Phase I motor program cannot be evoked by TrpA1-mediated activation. It could be that this manipulation fails to induce the correct pattern of activity in ETHRB-expressing neurons. Preliminary imaging results show that ETHRB-expressing neurons respond to ETH1 with oscillatory activity, and it is possible that these neurons directly couple to the Rk-expressing neurons through synaptic or electrical contacts and participate in generating Phase I behavior. However, further characterization of the activity of both the ETHRB- and non-CCAP/ETHRA neurons will be required to determine how they modulate VNC-Rk CPG activity (Diao, 2017).

Two input layer neurons that are common to the ETHRB- and non-CCAP/ETHRA groups express the major ecdysis neuromodulator, EH (Diao, 2015). The EH-expressing neurons, which are among the few cells to express both ETHRA and ETHRB, respond to ETH1 application at the onset of Phase II, and evidence from other insects indicates that EH targets CCAP-expressing neurons. EH is thus thought to be responsible for the release of CCAP and Bursicon, but this has not yet been verified in Drosophila where the EH receptor has yet to be identified. It was thus not possible to target EH receptor-expressing neurons in this study, but the identity and function of such neurons is likely to be critical to understanding the progression of the ecdysis sequence (Diao, 2017).

In general, it is worth noting that the neuromodulators regulating the ecdysis sequence are of the type called 'extrinsic,' because they are released from neurons that do not function in the circuits upon which they act. Extrinsic neuromodulatory neurons, however, must be components of the broader neural networks that generate behaviors, and the mechanisms that organize their activities are only beginning to be understood. In some cases, these mechanisms are surprising. For example, the neuromodulatory connections between neurons that govern two foraging states in C. elegans are orthogonal to the sensory-to-motor synaptic connections between these neurons, which are not involved in the state decision. There are currently few studies that jointly examine patterns of neuromodulatory and synaptic connectivity, and to understand how extrinsic neuromodulatory neurons integrate into the broader networks in which they function more examples of such networks are required. Elucidating the interactions of neurons in the input layer of the ecdysis network (in addition to interactions of the input layer with neurons in other layers) should provide insight into this general problem (Diao, 2017).

The finding that the motor output of the pupal ecdysis network is mediated by neurons that express the CCAP-R provides insight into the hitherto poorly understood mechanism of action of CCAP. This neuropeptide plays critical roles in the ecdysis of other insects, but genetic data demonstrate that in Drosophila it plays a subsidiary role to Bursicon, acting synergistically with that hormone to render pupal ecdysis more robust. The current results indicate that it does so by acting on motor neurons, and because CCAP is co-released with Bursicon from the ETHRA/CCAP neurons to govern the CPG transition at Phase II, this suggests a role for feed-forward signaling in the pupal ecdysis circuit (Diao, 2017).

Neuromodulatory feedforward pathways have been previously described and appear to be a common motif in motor network architectures. Feedforward loops of the type posited here for Bursicon and CCAP may be important in adjusting the coupling between Rk-expressing CPG neurons and their downstream motor neuron targets during Phase II. Compensatory adjustments in CPG, motor neuron and muscle activity by a single neuropeptide released from two different nodes in a feedforward loop have been described in the Aplysia feeding network where they guarantee stability of network output. Coordinating CPG activity with motor neuron activity may be particularly important for multifunctional CPGs, in which individual neurons participate in multiple motor patterns, as for example, in the leech swim/crawl network in which multifunctional neurons fire in phase with the contraction of one muscle group during swimming, but not necessarily during crawling (Diao, 2017).

The architecture of the pupal ecdysis network revealed in this study is similar to that of other motor circuits, such as those governing locomotion, feeding, and breathing in which higher order neurons modulate the activity of core CPGs to generate varied motor patterns. What is striking about neuromodulator action in the ecdysis circuit is its broad scope. ETH acts throughout the input layer to control different phases of pupal ecdysis behavior; Bursicon similarly regulates a large and essential set of neurons constituting the ecdysis CPG; and CCAP acts on many motor neurons necessary for generating the rhythmic ecdysis movements. The observation that Bursicon and CCAP signal from the input layer speaks to an organizational logic in which the ecdysial neuromodulators function together to provide coherence to the operation of the pupal ecdysis network by acting both within each hierarchical layer and by acting coordinately across layers. This organization is consistent with a generalized role for neuromodulatory systems in organizing neural activity to generate behavior (Diao, 2017).

The results also support the rationale of mapping neuromodulatory pathways as a strategy for identifying essential network circuits and their functional organization. It is worth noting that this mapping of the pupal ecdysis network was done without reference to patterns of synaptic connectivity. Synaptic connectomes have proved difficult to interpret, in part due to their dense interconnectivity. If, as has been previously emphasized, this interconnectivity reflects the multifunctionality of the underlying networks, and if the functional configuration of a network at any given time is determined by where and how neuromodulators are acting on its components, then patterns of neuromodulatory connectivity may provide a necessary complement to synaptic maps to render them interpretable. A key challenge will lie in identifying which neuromodulator systems play critical roles in establishing a network's output, but as the work here demonstrates, when these are known, the neuromodulatory connections can deliver substantial insight into how a neural network is organized (Diao, 2017).

A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila

The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. This study shows that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon alpha, which signals via its neuronal receptor DLgr2/Rickets. Bursicon alpha/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon alpha/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, this work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis (Scopelliti, 2018).

Maintaining systemic energy homeostasis is crucial for the physiology of all living organisms. A balanced equilibrium between anabolism and catabolism involves tightly coordinated signaling networks and the communication between multiple organs. Excess nutrients are stored in the liver and adipose tissue as glycogen and lipids, respectively. In times of high energy demand or low nutrient availability, nutrients are mobilized from storage tissues. Understanding how organs communicate to maintain systemic energy homeostasis is of critical importance, as its failure can result in severe metabolic disorders with life-threatening consequences (Scopelliti, 2018).

The intestine is a key endocrine tissue and central regulator of systemic energy homeostasis. Enteroendocrine (ee) cells secrete multiple hormones in response to the nutritional status of the organism and orchestrate systemic metabolic adaptation across tissues. Recent work reveals greater than expected diversity, plasticity, and sensing functions of ee cells. Nevertheless, how ee cells respond to different environmental challenges and how they coordinate systemic responses is unclear. A better understanding of ee cell biology will directly impact understanding of intestinal physiopathology, the regulation of systemic metabolism, and metabolic disorders (Scopelliti, 2018).

Functional studies on inter-organ communication are often challenging in mammalian systems, due to their complex genetics and physiology. The adult Drosophila midgut has emerged as an invaluable model system to address key aspects of systemic physiology, host-pathogen interactions, stem cell biology and metabolism, among other things. As in its mammalian counterpart, the Drosophila adult intestinal epithelium displays multiple subtypes of ee cells with largely unknown functions. Recent work has demonstrated nutrient-sensing roles of ee cells (Scopelliti, 2018 and references therein).

The role of Bursicon/DLgr2 signaling has long been restricted to insect development, where the heterodimeric form of the hormone Bursicon, made by α and β subunits, is produced by a subset of neurons within the CNS during the late pupal stage and released systemically to activate its receptor DLgr2 in peripheral tissues to drive post-molting sclerotization of the cuticle and wing expansion. A recent study demonstrated a post-developmental activity for the α subunit of Bursicon (Bursα), which is produced by a subpopulation of ee cells in the posterior midgut, where it paracrinally activates DLgr2 in the visceral muscle (VM) to maintain homeostatic intestinal stem cell (ISC) quiescence (Scopelliti, 2014; Scopelliti, 2016; Scopelliti, 2018).

This study reports an unprecedented systemic role for Bursα regulating adult energy homeostasis. This work identifies a novel gut/fat body axis, where ee cells orchestrate organismal metabolic homeostasis. Bursα is systemically secreted by ee cells in response to nutrient availability and acts through DLgr2+ neurons to repress adipokinetic hormone (AKH)/AKH receptor (AKHR) signaling within the fat body/adipose tissue to restrict the use of energy stores. Impairment of systemic Bursα/DLgr2 signaling results in exacerbated oxidative metabolism, strong lipodystrophy, and organismal hypersensitivity to nutrient deprivation. This work reveals a central role for ee cells in sensing organismal nutritional status and maintaining systemic metabolic homeostasis through coordination of an intestinal/neuronal/adipose tissue-signaling network (Scopelliti, 2018).

This study shows that ee cells secrete Bursα in the presence of plentiful nutrients, while caloric deprivation reduces its systemic release and consequently results in hormone accumulation within ee cells. Interestingly, it was observed that conditions leading to the latter scenario are accompanied by reduced bursα transcription. The reasons underlying the inverse correlation between midgut bursα mRNA and protein levels are unclear and may represent part of a negative feedback mechanism for ultimate control of further protein production. A similar phenomenon is described during the regulation of the secretion of other endocrine hormones, such as DILPs (Scopelliti, 2018).

The results show that Bursα within ee cells is preferably regulated in response to dietary sugars. This is further supported by the function of Glut1 as at least one of the transmembrane sugar transporters connecting nutrient availability to Bursα signaling. Glut1 is the closest homolog of the mammalian regulator of ee incretin secretion SLC2A2, and it has been shown to positively regulate the secretion of peptide hormones in flies (Park, 2014). Whether Glut1 is a central sensor of dietary sugars and hormone secretion by ee cells remains to be addressed. However, it is likely that, in the face of challenges, such as starvation, multiple mechanisms of nutrient sensing and transport converge to allow a robust organismal adaptation to stressful environmental conditions (Scopelliti, 2018).

Reduction of systemic Bursα/DLgr2 signaling induces a complex metabolic phenotype, characterized by lipodystrophy and hypoglycemia, which is accompanied by hyperphagia. These phenotypes are not due to poor nutrient absorption or uptake by tissues or impaired synthesis of energy stores but are rather a consequence of increased catabolism. This is supported by a higher rate of glucose-derived 13C incorporation into TCA cycle intermediates, accompanied by increased mitochondrial respiration and body-heat production (Scopelliti, 2018).

While glucose tracing experiments help explain the hypoglycemic phenotype of Bursα/DLgr2-compromised animals even in the context of hyperphagia, they do not directly address the reduction in fat body triacylglycerides (TAGs). The latter would require 13C6-palmitate tracing for assessment of the rate of lipid oxidation and incorporation into the TCA cycle. This was precluded by overall poor uptake of 13C6-palmitate into adult animals even after prolonged periods of feeding. However, the depletion of fat body TAG stores in the presence of normal de novo lipid synthesis in Bursα/DLgr2-impaired animals strongly suggests that at least part of the increased rate of O₂ consumption in those animals results from increased lipid breakdown via mitochondrial fatty acid oxidation. Consistently, increased O₂ consumption rates and the thermogenic phenotype of Bursα/DLgr2-deficient animals are attenuated upon reduction of AKH/AKHR signaling. Finally, the functional role of Hormone-sensitive lipase (dHSL) in the fat body further supports the regulation of lipid breakdown by AKH/AKHR signaling as at least one of the key aspects mediating the role of Bursα/DLgr2 signaling in adult metabolic homeostasis (Scopelliti, 2018).

Previous work revealed that ee Bursα is required to maintain homeostatic ISC quiescence in the adult Drosophila midgut; that is, in the midgut of unchallenged and well-fed animals (Scopelliti, 2014, Scopelliti, 2016). Such a role of Bursα is mediated by local or short-range signaling through DLgr2 expressed within the midgut VM (Scopelliti, 2014). This study demonstrates a systemic role of Bursα that does not involve VM-derived DLgr2 but rather signals through its neuronal receptor. In that regard, the paracrine and endocrine functions of Bursα/DLgr2 are uncoupled. However, the regulation of ee-derived Bursα by nutrients is likely to affect local as well as systemic Bursα/DLgr2 signaling. Retention of Bursα within ee as observed in conditions of starvation may impair the hormone's signaling into the VM, which, in principle, would lead to ISC hyperproliferation (Scopelliti, 2014). In fact, under full nutrient conditions, genetic manipulations impairing systemic Bursα signaling, such as ee Glut1 knockdown or osbp overexpression, lead to ISC hyperproliferation comparable with that observed upon bursα knockdown (Scopelliti, 2014). This represents an apparent conundrum, as ISC proliferation is not the expected scenario in the context of starvation. However, starvation completely overcomes ISC proliferation in Bursα-impaired midguts. This is consistent with recent evidence showing that restrictive nutrient conditions, such as the absence of dietary methionine or its derivative S-adenosyl methionine, impair ISC proliferation in the adult fly midgut, even in the presence of activated mitogenic signaling pathways (Obata, 2018). Altogether, these data support a scenario in which starvation, while preventing systemic and local Bursα/DLgr2 signaling, would not result in induction of ISC proliferation as a side effect (Scopelliti, 2018).

Drosophila DLgr2 is the ortholog of mammalian LGR4, -5, and -6 with closer homology to LGR4. While LGR5 and 6 are stem cell markers in several tissues, such as small intestine and skin, LGR4 depicts broader expression patterns and physiological functions. LGR4, -5, and -6 are best known to enhance canonical Wnt signaling through binding to R-spondins. However, several lines of evidence support a more promiscuous binding affinity for LGR4, which can act as a canonical G-protein coupled receptor inducing iCa2+ and cyclic AMP signaling (Scopelliti, 2018).

Interestingly, an activating variant of LGR4 (A750T) is linked to obesity in humans, while the nonsense mutation c.376C>T (p.R126X) is associated with reduced body weight. Recent reports show that LGR4 homozygous mutant (LGR4m/m) mice display reduced adiposity and are resistant to diet- or leptin-induced obesity. These phenotypes appear to derive from increased energy expenditure through white-to-brown fat conversion and are independent of Wnt signaling. The tissue and molecular mechanisms mediating this metabolic role of LGR4 remain unclear. Therefore, the current paradigm may lead to a better understanding of LGR4's contribution to metabolic homeostasis and disease. Importantly, the results highlight the intestine and ee cells in particular as central orchestrators of metabolic homeostasis and potential targets for the treatment of metabolic dysfunctions (Scopelliti, 2018).

Bursicon is an insect-specific hormone. Therefore, direct mammalian translation of the signaling system presented in this study is unlikely. However, given the clear parallels between the metabolic functions of DLgr2 and LGR4, analysis of enteroendocrine cell-secreted factors in mammalian systems may reveal new and unexpected ligands for LGR4 (Scopelliti, 2018).

REGULATION

Molecular characterization of Bursicon

The molecular analysis and cellular expression of the insect peptide neurohormone bursicon is described. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, the molecular weight of bursicon from different insects was determined to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, the Drosophila genome project was successfully searched for the gene encoding bursicon. With Drosophila as a tool, the function of the sequence has now been verified using transgenic flies. Sequence comparisons also allowed the verification that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution (Honegger, 2004).

Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster

At the last step of metamorphosis in Drosophila, the wing epidermal cells are removed by programmed cell death during the wing-spreading behavior after eclosion. The cell death is accompanied by DNA fragmentation demonstrated by the TUNEL assay. Transmission electron microscopy reveals that this cell death exhibits extensive vacuoles, indicative of autophagy. Ectopic expression of an anti-apoptotic gene, p35, inhibits the cell death, indicating the involvement of caspases. Neck ligation and hemolymph injection experiments demonstrate that the cell death is triggered by a hormonal factor secreted just after eclosion. The timing of the hormonal release implies that the hormone to trigger the death might be the insect tanning hormone, bursicon. This is supported by evidence that wing cell death is inhibited by a mutation of rickets, which encodes a G-protein coupled receptor in the glycoprotein hormone family that is a putative bursicon receptor. Furthermore, stimulation of components downstream of bursicon, such as a membrane permeable analog of cAMP, or ectopic expression of constitutively active forms of G proteins or PKA, induces precocious death. Conversely, cell death is inhibited in wing clones lacking G protein or PKA function. Thus, activation of the cAMP/PKA signaling pathway is required for transduction of the hormonal signal that induces wing epidermal cell death after eclosion (Kimura, 2004).

To determine whether a humoral signal coming from the head triggers cell death, the necks of flies were ligated at various times after eclosion and the wings were examined for cell death at 2 hours after the ligation. Ligation just after eclosion suppresses cell death and GFP was still detectable in the nuclei of wing epidermal cells after 2 hours. By contrast, when flies were ligated at 20 minutes after eclosion, the normal pattern of cell death was observed. Ligation at later stages correlates with an increased percentage of flies with wing epidermal cell death. Thus wing epidermal cell death is triggered by a signal emanating from the head shortly after eclosion (Kimura, 2004).

The cellular effects of cAMP are usually mediated by PKA. To determine whether this is also the case for wing epidermal cell death, the effect of reduction or elimination of PKA activity on cell death was examined. A dominant-negative form of the regulatory subunit of PKA (R*), whose ectopic expression is known to reduce the activity of endogenous PKA, was used. When R* was ectopically expressed using the en-Gal4 driver, many cells of the wings remained at 2 hours, or even at 8 hours, after wing spreading, resulting in separation between the ventral and dorsal cuticular sheets in the posterior compartment. Targeted expression of R* caused wavy or curly wings, probably due to the distortion between normal adhesion of dorsoventral cuticles in the anterior compartment and detachment of the cuticle in the posterior compartment (Kimura, 2004).

Next, DC0-dependent PKA activity was eliminated by generating clones of DC0 mutant cells within the developing wings. The DC0 gene in Drosophila encodes a catalytic subunit, one of the components of PKA. Since clones of DC0 mutant cells in the anterior compartment produce anterior duplication of the normal wing pattern, the clones in the posterior compartment were examined to investigate whether the death of wing epidermal cells marked with Histone-GFP is suppressed or not. The cells of the clones remained at 2 hours after wing spreading, although the surrounding cells had already been eliminated by cell death. Thus, reduction or elimination of PKA activity prevents the death of wing epidermal cells (Kimura, 2004).

The effects of constitutive activation of PKA on cell death were examined. A mutationally altered mouse catalytic subunit (mC*) was used that is resistant to inhibition by the regulatory subunit. The mutant catalytic subunit is constitutively active, irrespective of cAMP concentration, and can function in Drosophila cells. Using the en-Gal4 driver, the constitutively active catalytic subunit of mC* was expressed in wing epidermal cells. All eclosing flies had blistered wings. The wing epidermal cells died prior to wing spreading. Thus, constitutive activation of PKA causes the precocious death of wing epidermal cells (Kimura, 2004).

The induction of cell death was examined at various stages of pharate adults. As seen in the cases of cAMP injection and of ectopic expression of Gs{alpha}*, precocious cell death was induced at G stage and later. This indicates that wing cells acquire competence to respond to PKA activity by G stage, about 3 hours before eclosion (Kimura, 2004).

A mutation in the G-protein coupled receptor gene rickets inhibits wing epidermal cell death. In Drosophila, the rickets gene is a member of the glycoprotein hormone receptor family of the G-protein-coupled receptors and has been suggested to encode a bursicon receptor. Wing epidermal cell death, marked by Histone-GFP, was examined in rk mutants. In the mutants, wing epidermal cells remained at 2 hours, or even at 8 hours, after eclosion. To determine whether the inhibition of cell death is caused by a failure in the reception of a hormonal signal inducing death, the effects were examined of 8-Br-cAMP and hemolymph injection into rk mutants that were neck-ligated at eclosion. In wild-type flies, injection of hemolymph from wild-type flies at 30 minutes after eclosion and injection of 8-Br-cAMP induces cell death. However, in rk mutants, cell death was induced by injection of 8-Br-cAMP but not by injection of hemolymph. This indicates that the mutant cells could not receive the hormonal signal in the hemolymph, although the activation of cell death by cAMP/PKA signaling was normal in the mutant cells (Kimura, 2004).

The peptide hormone, Bursicon, is known to play a role in the post-ecdysial phase of development. Bursicon has been shown to be released before wing expansion and to hasten the tanning reaction, serving to harden the newly expanded cuticle. The results of this study suggest that the hormone that induces cell death of the wing epidermis could be bursicon. (1) Neck ligation and hemolymph injection experiments have demonstrated that the triggering signal to induce death is a humoral factor released after eclosion. This temporal pattern of death-inducing activity in the hemolymph corresponds to that of bursicon. (2) Injection of cAMP induced cell death, implicating cAMP as the second messenger in the cell death pathway. Studies in blowflies have shown that bursicon also acts through cAMP. Recently, in Drosophila, cAMP was shown to induce cuticular melanization in a fashion similar to bursicon. (3) Reception of the hormonal signal inducing cell death is mediated by a probable bursicon receptor, Rickets (DLGR2), which also acts through cAMP. (4) In Lucilia cuprina, it has been proposed that bursicon is the same as fragment disaggregating hormone, which increases the circulating filamentous cellular fragments derived from post-ecdysial death of the wing epidermal cells. Taken together, it is likely that bursicon coordinates events such as the cell death of wing epidermis and the subsequent tanning and hardening of the cuticle. However, another possibility cannot be ruled out -- namely, that several humoral factors could signal through the pathway. Identification of a bursicon gene as CG13419 in Drosophila will facilitate genetic approaches to understand the role of bursicon in wing epidermal cell death (Kimura, 2004).

Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2

All arthropods periodically molt to replace their exoskeleton (cuticle). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Bursicon, the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The Pburs/Burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G protein-coupled receptor DLGR2 (Rickets), leading to the stimulation of cAMP signaling in vitro and tanning in neck-ligated blowflies. Native bursicon from Periplaneta americana is also a heterodimer. In D. melanogaster the levels of pburs, burs, and DLGR2 transcripts are increased before ecdysis, consistent with their roles in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species have revealed the colocalization of pburs- and burs-immunoreactivity in some of the neurosecretory neurons that also express crustacean cardioactive peptide. Forty-three years after its initial description, the elucidation of the molecular identity of bursicon and the verification of its receptor allow for studies of bursicon actions in regulating cuticle tanning, wing expansion, and as yet unknown functions. Because bursicon subunit genes are homologous to the vertebrate bone morphogenetic protein antagonists, these findings also facilitate investigation on the function of these proteins during vertebrate development (Luo, 2005).

Based on GenBank searches for novel cystine knot proteins as potential dimerization partners for burs, the CG15284 gene was identified and named partner of burs (pburs). The predicted Pbursprotein is highly conserved among D. melanogaster, mosquito (Anopheles gambiae), honey bee (Apis mellifera), and silkworm (Bombyx mori), and all contain the 11 cysteine residues that are also present in the D. melanogaster burs. A stretch of four residues in D. melanogaster Pburs is identical to a partial peptide of purified bursicon found in P. americana. Based on the known cystine knot structures of TGF-beta and CG-beta, the structure of Pburs could be predicted. Both Pburs and Burs are likely to form a cystine knot and contain two intramolecular disulfide bonds between C2-C8 and C5-C11. In addition, a free cysteine at position 6 may be involved in the formation of an intermolecular bridge. Analyses of the pburs gene structures from the four insects indicate that they consist of two or three exons. Of interest, the first exon in D. melanogaster is split into two exons in A. mellifera and B. mori, whereas exons 2 and 3 in A. mellifera and B. mori are combined into one exon in A. gambiae. Phylogenetic analyses based on the cystine knot region of several related proteins further indicate that Pburs, together with Burs, show close sequence relationships with cystine knot proteins in the human BMP antagonist family. Some of these proteins have been found to antagonize the actions of BMP ligands during embryonic development and organogenesis (Luo, 2005).

To assess receptor-binding ability, recombinant epitope-tagged Bursicon was prepared by using two sequential affinity columns against polyHis and the FLAG epitope appended to Burs and Pburs, respectively. The purity of the Bursicon heterodimer was confirmed by using SDS/PAGE followed by Coomassie blue staining. Under nonreducing conditions, purified Bursicon migrates ~38 kDa, whereas two lower bands are evident under reducing conditions and confirmed to be Pburs and Burs monomers by using specific antibodies. Purified Bursicon was iodinated and found to bind to cells expressing DLGR2. ¹²⁵I-bursicon binding was displaced in a dose-dependent manner by nonlabeled bursicon but was not affected by Pburs or Burs alone. Conversion of the displacement curve to the saturation and Scatchard plots indicated that Bursicon binds to DLGR2 with high affinity (Luo, 2005).

To examine the expression of pburs, burs, and DLGR2, real-time PCR analyses was performed by using RNA preparations from different developmental stages of D. melanogaster. The transcript levels for pburs and burs show a similar pattern of regulation. They were both low in larva (stages 1 and 2) and gradually increase in pupal stages (3 and 4) before reaching the highest levels in pharate adults (stage 7), in preparation for tanning after adult eclosion. For DLGR2 transcripts, an increase was found in early pupae (stage 4), followed by a decrease in the late pupae (stage 6), before increasing dramatically in pharate adults. In the adult animals (stage 8), all three transcripts are low (Luo, 2005).

Because the sequences of both Pburs and Burs are highly conserved in insects, antibodies against D. melanogaster Pburs or Burs were used to detect neurons expressing these proteins in the central nervous systems of three different insect species. Immunohistochemical staining for Pburs and Burs in the third-instar larva of D. melanogaster revealed that Pburs was coexpressed with Burs in four bilateral neurons in thoracic and abdominal neuromeres of the ventral nervous system. However, the somata in the subesophageal and posterior abdominal neuromeres expressed only Burs. Likewise, colocalization of Pburs and Burs occurred in the large bilateral lateral neurosecretory neurons of the first three unfused abdominal ganglia and in all anterior bilateral cell pairs in the thoracic ganglia of P. americana and in homologous neurons in the thoracic and abdominal ganglia of the cricket Teleogryllus commodus. In these somata, the immunostaining was found in large granules that characterize these endocrine cells (Luo, 2005).

Further, by using antibodies against Pburs and CCAP in P. americana and in situ hybridization of pburs in wild-type and CCAP neuron-ablated fly mutants, the data suggest that Pburs, like Burs, is colocalized with CCAP in some CCAP-immunoreactive neurons (Luo, 2005).

In D. melanogaster, mutations in the rickets gene show crossed postscutellar bristles and kinked femurs (Edmondson (1948). These mutant flies carry lesions in the DLGR2 gene and fail to initiate tanning and wing expansion after adult emergence. Although rickets mutants do not melanize when injected with ganglia extracts containing bursicon, they do than in response to injection of an analog of cAMP (Baker, 2002), consistent with the findings that in cells transfected with DLGR2 bursicon stimulates cAMP production. Because flies with burs mutations show the same phenotypes as rickets mutants, burs was proposed to encode a ligand for DLGR2. The present findings demonstrate, however, that Burs needs Pburs as a partner for DLGR2 activation (Luo, 2005).

In addition to the induction of cuticle tanning, bursicon also has been implicated in a stereotyped behavioral program for wing expansion and in the postecdysial cell death of wing epidermal cells. Real-time PCR results showing increases of pburs, burs, and DLGR2 transcripts at pupariation and eclosion stages of D. melanogaster are consistent with the importance of these genes during insect metamorphosis (Luo, 2005).

In D. melanogaster, three LGR genes have been identified. They encode seven transmembrane proteins with a large ectodomain containing leucine-rich repeats. These three fly LGRs show sequence homology with the three subgroups of mammalian LGRs, including the group A glycoprotein hormone receptors, group B LGR4/5/6, and group C LGR7 and -8. Fly DLGR2, like mammalian LGR4/5/6, has 17-18 leucine-rich repeats, likely involved in ligand binding. A recent study identified a group B LGR ortholog in the gastropod Crassostrea gigas (oyster), suggesting that these receptors have ancient origins. Although the ligands for LGR7 and LGR8 recently have been identified, the ligands for the group B vertebrate LGRs are unknown. The insect pburs and burs genes are homologous to the vertebrate BMP antagonist family of genes found to be important during embryonic development and organogenesis. Only some members of the BMP antagonist family are competitive antagonists capable of direct binding to BMPs, and the exact mechanisms of actions of most proteins in this family are still unknown. Future studies will reveal whether these cystine knot proteins are ligands for vertebrate orphans LGR4/5/6. Of interest, recent studies have demonstrated that mutations in the LGR4 and LGR5 genes in mice are associated with perinatal lethality, suggesting important roles during embryonic development (Luo, 2005 and references therein).

The identification of two cystine knot polypeptides as subunits for the heterodimeric bursicon in insects, together with the demonstration of specific binding and activation of a G protein-coupled receptor with leucine-rich repeats, finally allow for an investigation of the diverse aspects of the physiological functions of this important neuroendocrine hormone (Luo, 2005).

Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish

Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon β (burs β), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies have show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown. This report shows in Drosophila that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs), Diptericin (Dpt), Cecropin B (Cec B), Attacin A (Att A), Turandot B (Tot B), and Attacin B (Att B), but not Drosomycin (Drs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. These results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data it is inferred that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle (An, 2012).

The data in this paper support the hypothesis that the CNS influences innate immunity via secretion of a neurohormone and thus expands the biological roles of bursicon beyond cuticle tanning and wing expansion. Several points are germane. First, the expression patterns of genes encoding burs alpha and burs β subunits and six AMPs in untreated pharate and newly-eclosed adults appear in strong inverse correlation. Second, injection of r-burs α/α or burs β/β homodimers to the 24 h-old files, which displayed low levels of bursicon transcripts and AMP genes, up-regulates AMP genes, demonstrating a role for bursicon homodimers in mediating AMP gene transcription in vivo. Third, bursicon acts in the novel homodimer configuration. Fourth, the bursicon homodimers induce expression of genes encoding AMPs via the activation of the NF-kappaB transcription factor Relish. And fifth, burs α/α or burs β/β homodimers do not appear to regulate their effects on the immune system via the established heterodimer receptor, DLGR2. Sequence analysis revealed that the burs α and β subunits have no similarity to bacterial cell wall proteins, which bind the peptidoglycan recognition protein (PGRP) to activate immune responses. Although PGRP binding to bursicon has not been experimentally ruled out, it is inferred that bursicon homodimers do not act through the PGRP. Hence, bursicon homodimers activate components in the Imd signaling pathway, downstream of PGRP, but upstream of Relish. Future studies will focus on the identification of the novel receptor(s) involved in the action of bursicon homodimers on the immune system. Despite the preponderance of work on mammalian immunity, exactly how the CNS controls inflammation and the immune response is not understood completely. Perhaps the Drosophila model with its abundant genetic repertoire will help solve this ancient problem(An, 2012).

Bursicon is a member of the cystine knot protein family, which includes vertebrate glycoprotein hormones, growth factors, mucins, and bone morphogenetic protein antagonists. All these hormones, including bursicon, share a common structural feature of a α-subunit and a β-subunit which form the physiologically operational heterodimers. Some, such as the placental chorionic gonadotropin, also form homodimers to execute a different physiological function. It has been shown that each of the bursicon subunits forms a homodimer, shown in vitro in Drosophila by Western blot and in vivo in several insect species including Drosophila by immunocytochemistry, but the function of the homodimers was, until now, unknown. This study identified one role for the bursicon homodimers as mediators of the prophylactic expression of genes encoding AMPs(An, 2012).

It is also noted that bursicon homodimers induce expression of several turandot and Tep1 genes, markers for the JAK/STAT pathway. Since the transcriptional regulation of the JAK/STAT pathway requires inputs from the Imd pathway, up-regulation of turandot and Tep1 genes could result from up-regulation of the Imd pathway. The biological significance of these gene products extends beyond anti-microbial actions to the generalized responses to extreme stressors (Ekengren, 2001a). While other stress-responsive proteins, such as heat shock proteins, act within cells, the Turandot proteins are secreted into the hemolymph following a variety of stress experiences (Ekengren, 2001b). Like developmental and reproductive events in many animals, molting produces actual and potential stresses in insects, including increased energy demands (producing reactive oxygen species), water loss, ion imbalances, injury and infection. It is concluded that the expression of general stress-responsive genes could be an important adaption during the highly susceptible time of the molting cycle (An, 2012).

Targeted inactivation of the rickets receptor in muscle compromises Drosophila viability

Bursicon is a hormone that modulates wing expansion, cuticle hardening, and melanization in Drosophila melanogaster. Bursicon activity is mediated through its cognate G protein-coupled receptor, Rickets. This study developed a membrane tethered Bursicon construct that enables spatial modulation of Rickets mediated physiology in transgenic flies. Ubiquitous expression of tethered Bursicon throughout development results in arrest at the pupal stage. The few organisms that eclose fail to undergo wing expansion. These phenotypes suggest that expression of tethered Bursicon inhibits Rickets mediated function. Consistent with this hypothesis, this study showed in vitro that sustained stimulation of Rickets by tethered Bursicon leads to receptor desensitization. Furthermore, tissue specific expression of the tethered Bursicon inhibitor unraveled a critical role for Rickets in a subset of adult muscles. Taken together, these finds highlight the utility of membrane tethered inhibitors as important genetic/pharmacological tools to dissect the tissue specific roles of GPCRs in vivo (Harwood, 2014).

Bursicon is a heterodimeric cystine-knot protein required for wing expansion and cuticle hardening in a variety of insects. Early studies performed in blowflies and cockroaches, showed a hormone of unknown molecular identity released from the central nervous system was important for tanning and wing expansion. More than four decades later the molecular identity of Bursicon and its cognate receptor, rickets were subsequently identified in Drosophila melanogaster. Rickets (Rk or dLGR2) is a member of the leucine rich repeat-containing subfamily of G protein coupled receptors (GPCRs) which is expressed both in the CNS and in the periphery. Previous studies have shown that a membrane anchored single subunit fusion construct of the Bursicon heterodimer (CFP-tBur-&betal-α) can activate Rk in vitro in a concentration dependent manner. Membrane tethered ligands (MTLs) are cDNA constructs that express genetically encoded peptide hormones anchored to a transmembrane domain via a protein linker. The generation of this complex Bursicon heterodimeric tethered construct was done as part of an ongoing effort to sequentially develop a broad range of MTLs that selectively activate either insect or mammalian GPCRs (Harwood, 2014).

A major advantage of using MTL technology in vivo is that it enables activation of receptors in a targeted tissue without the confounding effects of soluble ligand diffusion. The transgenic Gal4/UAS system in conjunction with membrane tethered Bursicon offer an excellent model system in which to better understand the tissue dependence of Rk mediated signaling. In Drosophila, wing expansion occurs within 1 hour following eclosion. A tightly choreographed motor program is required for wing expansion and cuticle hardening. This series of events coincides with a biphasic release of Bursicon from a subset of crustacean cardioactive peptide (CCAP) positive neurosecretory cells. Bursicon is first released from the subesphogeal ganglion, followed by secretion from the abdominal ganglion into the hemolymph. The Drosophila circulatory system then disperses Bursicon throughout the organism. While most studies have focused on the location of Bursicon release, much less research has addressed the importance of tissue selective Rk activation (Harwood, 2014 and references therein).

Previously it was thought that Rk was only required following eclosion, as a trigger for wing expansion and cuticle hardening. This postulate was based on data from two fly stocks, rk1 and rk4 which were thought to be receptor nulls. However a more recent study has shown that these flies are hypomorphs. In fact global knockdown of rk in vivo using RNAi results in developmental arrest, rather than just impaired wing expansion, melanization, and cuticle hardening. A recent study also showed that deletion of the Bursicon β subunit results in significant lethality throughout pupariation, specifically during ecdysis. There has been no comprehensive study which has specifically examined what tissues require rk for proper development. A previous study which utilized a GFP reporter, demonstrated rk expression in the epidermis. More detailed analysis of rk transcript levels as revealed by Fly Atlas and RNA-Seq studies have shown that this receptor is expressed at low levels throughout development. However, which tissues or cells require rk expression for proper development remains an unexplored area of inquiry (Harwood, 2014).

Use of the Bursicon MTL, CFP-tBur-&betal-α), offered a novel approach to investigate the tissue specific requirements of rk. The use of tissue specific Gal4 drivers to target expression of the fused heterodimer provided a means to selectively modulate rk without the confounding effects of soluble ligand diffusion. Parallel studies using rk RNAi transgenic flies enabled a complementary approach to confirm conclusions drawn through the use of the membrane tethered ligand (Harwood, 2014).

Prior investigations have shown that a membrane tethered agonist can trigger long term receptor activation in flies. In this current study, it is illustrated that there is an alternative potential consequence that may result from membrane tethered ligand expression in vivo. Specifically the Bursicon MTL triggers Rk desensitization resulting in functional blockade of the receptor. This mechanism is supported by in vitro data demonstrating that long term Bursicon stimulation, using either soluble ligand or the corresponding MTL, essentially eliminated further Bursicon mediated signaling. This study shows that ubiquitous receptor inactivation using the CFP-tBur-&betal-α) in vivo results in developmental lethality. Furthermore, by expressing membrane tethered Bursicon with a collection of increasingly focused tissue specific Gal4 drivers, it was possible to show an essential role during eclosion and subsequent wing expansion. Parallel studies with rk RNAi constructs support each of the above conclusions (Harwood, 2014).

This study utilized a membrane tethered Bursicon construct (CFP-tBur-&betal-α) o probe the role of rk in development. Previous studies have shown that when assessed in vitro; CFP-tBur-&betal-α is an rk agonist (Harwood, 2013). Initial in vivo investigations revealed that expression of CFP-tBur-&betal-α in otherwise rk/Bursicon wildtype Drosophila (W1118) led to lethality and wing expansion defects, both unanticipated phenotypes. Although it is difficult to predict the effect of hormonal imbalance in vivo, it was hypothesized that the agonist activity of CFP-tBur-&betal-α would provide a gain of function and would be useful to rescue Bursicon mutant fly lines. Contrary to this expectation, the phenotypes resulting from ubiquitous expression of CFP-tBur-&betal-α were loss of function (i.e. lethality and wing expansion defects) and resembled those observed with knockdown of rk using RNAi. Flies that expressed CFP-tBur-&betal-α or rk RNAi ubiquitously generally reached pupal development however failed to eclose. Dead flies were fully developed but did not emerge from their pupal cases. Based on the parallel phenotypes with the RNAi flies, it was hypothesized that expression of CFP249 tBur-&betal-α resulted in a decrease of rk mediated signaling (Harwood, 2014).

Prior pharmacological studies have shown that sustained activation of a GPCR can trigger receptor desensitization, in turn leading to decreased receptor mediated signaling. Based on this precedent, it was postulated that CFP-tBur-&betal-α expression induces chronic stimulation of Rk in vivo resulting in receptor desensitization. The desensitized Rk receptor may not adequately respond to endogenous Bursicon at key developmental stages. To examine this possibility, in vitro studies were done comparing the effects of CFP-tBur-&betal-α and soluble Bursicon conditioned media on Rk activation/desensitization. Results show that long term stimulation of Rk (overnight) with either soluble or membrane tethered Bursicon renders the receptor unable to further respond to Bursicon. Notably, the desensitization of Rk is receptor specific. HEK 293 cells expressing recombinant Rk that has been desensitized to Bursicon can still signal in response to β2agonist treatment via endogenously expressed β2AR, which also signals through Gsα (Harwood, 2014).

Desensitization has previously been documented among other LGR receptors. For example, the human receptor LGR5 is known to be constitutively internalized and is one of the most evolutionarily related orthologs to rk. Notably, the C-terminal region of both Rk and LGR5 includes multiple serine residues, which, in the case of LGR5 has been shown to play an important role in desensitization. Taken together, it is proposed that tethered Bursicon mediated desensitization of Rk may underlie the loss of function phenotypes observed in vivo. Transgenic flies expressing tethered Bursicon under a UAS inducible promoter provide an important complementary genetic tool for studying the rickets Bursicon system (Harwood, 2014).

This study has shown that ubiquitous expression of CFP-tBur- β-α leads to developmental arrest at the pupal stage. Escapers that survive to adulthood show melanization and wing expansion phenotypes, features characteristic of rk knockdown. CFP-tBur- β-α induces phenotypes that are readily monitored, and provides a useful tool to further dissect the role of rk in the developing fly (complementing rk RNAi constructs) (Harwood, 2014).

During a previous screen performed in the Kopin lab, it was noted that RNAi mediated knockdown of the heterotrimeric G protein subunit Gαs, with muscle specific drivers resulted in developmental arrest (personal communication of Isabelle Draper to Harwood, 2014). It was hypothesized that Rk may be the receptor upstream of Gαs in muscle which led to this phenotype. To assess this possibility, CFP-tBur- &betal-α expression was restricted to muscle under the control of the pan mesodermal driver HOW-Gal4. CFP-tBur-&betal-α expression resulted in defects that phenocopied those seen with both ubiquitous expression of the tethered ligand and Gαs knockdown in muscle (i.e. lethality, wing phenotypes). Similar phenotypes were also observed when rk was downregulated using rk RNAi, confirming that the receptor was important in muscle. A series of Gal4 driver lines was used to target the tethered ligand to selected tissues and thus define the critical cell type(s) that require rk and underlie the lethality/wing expansion phenotypes. Although many studies have focused on the spatial and temporal release of Bursicon, the localization of the Bursicon receptors that are essential for survival, has remained elusive. This study strongly supports that a peripheral Rk, localized in muscle, plays an important role in Drosophila development (Harwood, 2014).

Expression of either CFP-tBur-&betal-α, or rk RNAi, in muscle leads to comparable lethality/wing phenotypes indicating that the receptor is present postsynaptically. In contrast, expression of rk RNAi in motor neurons (using the D42-Gal4 driver) has no effect, suggesting the absence of a presynaptic receptor. Expression of CFP-tBur- &betal-α using the same driver however leads to a wing defect phenotype. By design, CFP-tBur- &betal-α) anchors in the membrane and projects into the extracellular space (Harwood, 2013). Its function is defined primarily by three structural elements: transmembrane domain, linker, and peptide. It is postulated that tethered Bursicon anchored in the motor neuron may act on muscle Rk in trans leading to partial desensitization of the receptor thus inducing the wing expansion defect. In addition to defining a role for rk in muscle tissue, the role of rk during myogenesis was examined. It is well-established that during metamorphosis, most of the adult muscles are formed de novo from progenitor cells, i.e. adult muscle precursors (AMPs) located on the larval wing imaginal disc and leg imaginal disc. Undifferentiated AMPs express high levels of the helix-loop-helix transcription factor, Twist. As twist is downregulated, myoblasts commit to the muscle lineage and differentiate. This occurs in conjunction with an interplay of many transcription factors. A core regulatory network includes Tinman (cardiac muscle specification), Myocyte Enhancer Factor-2, Mef2 (which plays a key role in myoblast fusion/ formation of somatic muscles) and Apterous (which specifies selected subtypes of somatic muscles). Based on the above, selected drivers were used to target rickets at different stages of the myogenic process using either CFP-tBur- &betal-α or rk RNAi. Using the twist-gal4 driver this study has shown that downregulation of rk in AMPs had no effect on survival or wing expansion. This suggests that very early myogenesis is not altered by the absence of rk. In contrast it was shown that altering rk in differentiating myotubes (Mef2 positive cells) compromised survival and wing expansion (Harwood, 2014).

Bursicon and CCAP are co-packaged and released directly on to heart muscle. CCAP has an important role in modulating heart function. Given the coordinated synthesis and release of these two hormones, it was asked whether Rk mediated eclosion may also be linked to receptors expressed on Drosophila heart muscle. The analysis ruled out this possibility; decreased rk function in cardiac muscle did not compromise either eclosion or wing expansion (Harwood, 2014).

Whether expression of rk in selected subtypes of pharate adult muscles could account for the observed CFP-tBur-&betal-α/rk RNAi induced phenotypes was investigated. For these studies, the Act88F-Gal4 driver line was used that targets major muscles in the adult fly, including the indirect flight muscles. Expression of CFP-tBur-β-α or knockdown of rk in adult muscle results in developmental lethality prior to eclosion while escaper flies fail to expand their wings. Comparison of the tissues tissues that are targeted by the apterous Gal4 and Act88F Gal4 enabled exclusion of selected muscle subtypes potentially underlying the rk mediated phenotypes. Actin 88F is a muscle actin predominantly expressed in the thoracic indirect flight muscles (IFMs). Notably, apterous is absent in the IFMs, making this an unlikely target. Actin88F is also expressed in the mesothoracic leg (tibial depressor muscles), as well as in the abdominal muscles (both ventral and dorsal). These two muscle types thus emerge as potentially important for rk signaling-regulation of eclosion (Harwood, 2014).

It is well established that both leg and abdominal muscles are important during metamorphosis. At 12 hours after puparium formation, contraction of the abdominal muscles forces an air bubble forward which in turn, triggers head eversion. In addition, at the end of the pupal stage, the newly formed adult uses its legs to free itself from the case. Consistent with the hypothesis that rk is present in leg muscle, Bursicon immunoreactive neurons have been shown to directly innervate the leg of the cricket, Gryllus bimaculatus. The potential role of rk in leg muscle could also explain the kinked leg phenotype observed with rk classical mutants (Harwood, 2014 and references therein).

In conclusion, a membrane tethered ligand (CFP-tBur-&betal-α) was developed that negatively regulates rk function when expressed in vivo. Using this novel tool in conjunction with existing RNAi fly lines adult muscle were identified as a tissue that requires rk expression for survival and wing expansion in Drosophila. In particular, based on this analysis it appears leg and abdominal muscles, or a subset of these, could be important. These studies set the stage for future investigations aimed at further understanding the role of muscle rk in fly development (Harwood, 2014).

Membrane tethered Bursicon constructs as heterodimeric modulators of the Drosophila G protein-coupled receptor rickets

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, this study explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila Bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor Rickets (Rk). Rk/Bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. Two distinct MTL constructs were engineered, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or β Bursicon subunit. Coexpression of the two complementary Bursicon MTLs triggered Rk-mediated signaling in vitro. Functionally active Bursicon MTLs were generated in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-β or β-α. Carboxy-terminal deletion of 32 amino acids in the β-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble Bursicon. This study has thus generated membrane-anchored Bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of Bursicon in the genetically tractable Drosophila model organism. In addition, this success in generating functionally diverse Bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins (Harwood, 2013).

Previously, only MTLs that included short peptide ligands (up to 39 amino acids) have been described. In contrast, the mature Bursicon subunits, α and β, are 141 and 121 amino acids, respectively. Furthermore, each of these subunits is a cystine-knot protein that includes a series of intramolecular disulfide bridges which confer tertiary structure. As an additional prerequisite of agonist activity, the α and β subunits must interact to form a structurally integrated heterodimer (Harwood, 2013).

Given the stringent requirements underlying the formation of active soluble Bursicon, including cellular coexpression, coprocessing, and cosecretion, the success in generating corresponding functional membrane tethered ligands could not have been anticipated. Initially, it was demonstrated that expression of both single tethered Bursicon subunits (α and β) in the same cell was sufficient to generate an active ligand. Follow-up studies revealed that coexpression of soluble and tethered complementary subunits also enabled the formation of active ligand. In contrast, when a single soluble subunit was added as conditioned media to cells expressing a tethered complementary subunit, no agonist activity was detectable. This finding suggests that intracellular assembly of the α-β heterodimer is a critical step in the formation of active hormone. These observations are consistent with reports on the heterodimerization requirements of soluble Bursicon and other cystine-knot proteins that are known to undergo intracellular assembly prior to secretion as an active ligand. Remarkably, both membrane tethered and soluble Bursicon subunits, despite the complexity of processing, appear to be fully compatible with each other in forming active heterodimers (Harwood, 2013).

In an attempt to further understand the structural requirements underlying tethered Bursicon function, constructs were generated in which both the α and β subunits were included in a single MTL. Since an active tethered ligand can be generated as either a β-α or α-β fusion construct, neither a free N nor a free C terminus is a requirement for agonist activity. It is noteworthy that conditioned medium containing a soluble form of the Bursicon fusion protein tested in the β-α arrangement also shows agonist activity. Whether tethered or soluble, the Bursicon fusions are active ligands. Observations with Bursicon reveal another parallel with mammalian heterodimeric cystine-knot proteins. Fusion of the α and β subunits of mammalian glycohormones including TSH, LH, and FSH as single soluble peptides also results in ligands that can activate their corresponding mammalian GPCR (Harwood, 2013).

The generation of tethered Bursicon fusion proteins provided a simplified model system to define domains of the dimer that are important for agonist activity. These experiments were guided by prior observations that the β subunit of mammalian glycohormones provides specificity and affinity for cognate receptors, whereas the α subunit is required for receptor activation. Furthermore, the literature suggests that the C terminal domain of the glycohormone α subunit is an important determinant for ligand activity. Based on this knowledge, a series of deletions were generated in the C terminus of Burs α in the context of the tBur-β-α heterodimer. These experiments demonstrated that the C terminal domain in tethered Bursicon was essential for Rk activation. One of the deletion mutants in which 32 C-terminal residues were truncated (designated as Δ32) not only led to loss of agonist activity, but also markedly inhibited the function of soluble Bursicon. This observation suggests that, once a domain essential for agonist activity is removed in the corresponding MTL, the remaining truncated peptide can inhibit soluble agonist–induced signaling. However, an MTL with a larger C terminal deletion (Δ35), although also lacking agonist activity, was much less effective (versus Δ32) in blocking soluble Bursicon–induced signaling. The difference between Δ32 and Δ35 is that three additional highly conserved residues including a critical cysteine are truncated in Δ35. The loss of these three residues may have compromised the tertiary structure of the tethered ligand, in turn explaining the functional difference in constructs. Soluble versions of the Δ32 and Δ35 constructs did not confer the same ability to block ligand-induced signaling. Thus, it is possible that membrane anchoring is required to generate a functional antagonist (Harwood, 2013).

It is of note that the GPCR targeted MTLs that had been reported prior to this study all shared a common orientation, in which the peptide ligand was expressed with a free extracellular N terminus. In contrast, the Bursicon MTLs were engineered with the opposite orientation (i.e., with a free extracellular carboxy terminus). This was achieved by incorporating a different transmembrane domain anchor (a type II TMD) into the construct. The ability to generate membrane-tethered ligands in either orientation markedly enhances the potential utility of MTL technology. For many peptides, orientation may be a critical factor in generating an active MTL. It is well established that, for peptide hormones recognizing class B GPCRs (e.g., secretin, parathyroid hormone, corticotropin releasing factor, glucagon-like peptide-1, gastric inhibitory polypeptide), the critical determinants of ligand efficacy reside in the N terminal domain of the hormone. Previous studies have shown that each of these peptides remains active when incorporated into an MTL that includes a type I TMD, i.e., the extracellular free end of the peptide is the N terminus. In contrast, peptide ligands recognizing class A GPCRs are more diverse. As examples, the amino termini of chemokines are generally considered critical for ligand activity, whereas for neuropeptides, functional determinants are often localized at the carboxyl terminus. In the latter case, it is anticipated that MTLs including a type II TMD will preserve biologic activity when corresponding peptides are anchored to the cell membrane (Harwood, 2013).

In summary, a strategy has been developed that can be widely applied to the study of peptide ligands. More specifically, Bursicon MTLs were identified that either activate or block Rk-mediated signaling. These findings set the stage for future in vivo studies. In the investigations to follow, tethered constructs will be selectively expressed in targeted tissues of Drosophila, thus exploring the utility of the approach for defining corresponding Rk-mediated pathways/physiologies. Precedent with these Bursicon MTLs will set the stage for parallel studies using other tethered cystine-knot proteins as tissue selective molecular probes. Candidate MTLs include mammalian glycohormones as well as non-GPCR regulators such as bone morphogenetic protein antagonists. The efficiency and flexibility of recombinant MTL technology will enable generation of a wide range of unique tools to complement the use of soluble ligands in understanding corresponding receptor-mediated physiologies (Harwood, 2013).

Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death

Hormones are often responsible for synchronizing somatic physiological changes with changes in behavior. Ecdysis (i.e., the shedding of the exoskeleton) in insects has served as a useful model for elucidating the molecular and cellular mechanisms of this synchronization, and has provided numerous insights into the hormonal coordination of body and behavior. An example in which the mechanisms have remained enigmatic is the neurohormone Bursicon, which, after the final molt, coordinates the plasticization and tanning of the initially folded wings with behaviors that drive wing expansion. The somatic effects of the hormone are governed by Bursicon that is released into the blood from neurons in the abdominal ganglion (the B_AG), which die after wing expansion. How Bursicon induces the behavioral programs required for wing expansion, however, has remained unknown. This study shows by targeted suppression of excitability that a pair of Bursicon-immunoreactive neurons distinct from the B_AG and located within the subesophageal ganglion in Drosophila (the B_SEG) is involved in controlling wing expansion behaviors. Unlike the B_AG, the B_SEG arborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the B_AG. Indeed, it was shown that animals lacking Bursicon receptor function have deficits both in the humoral release of Bursicon and in posteclosion apoptosis of the B_AG. These results reveal novel neuromodulatory functions for Bursicon and support the hypothesis that the B_SEG are essential for orchestrating both the behavioral and somatic processes underlying wing expansion (Peabody, 2008).

The study of insect ecdysis has provided a productive model for investigating the hormonal control of behavior. Bursicon acts directly after adult ecdysis in Drosophila to initiate the somatic processes and motor programs underlying wing expansion and to induce rapid cuticle tanning. In Drosophila,the architecture of Bursicon release differs from that of the other principal hormones that regulate ecdysis-related behaviors in that it is secreted not from a homogenous group of cells subject to common regulatory control, but instead from two distinct subsets of neurons. The work presented in this study demonstrates that this anatomical partition mirrors functional differences between the two subsets of Bursicon-expressing neurons. One group (the B_AG) secretes Bursicon into the hemolymph, and the other (the B_SEG) releases it widely in the nervous system to initiate the motor programs that underlie wing expansion. The observation that secretion of Bursicon from the B_AG is impaired in rickets mutants is consistent with a model in which Bursicon secreted by the B_SEG also modulates release from the B_AG. These results thus provide a framework for understanding how the somatic actions of Bursicon are coordinated with its effects on behavior. In addition, the finding that apoptosis of the B_AG is delayed in rickets mutants exposes a novel neuromodulatory function for Bursicon (Peabody, 2008).

That Bursicon is released into the blood from neurons in the abdominal ganglia has long been known from work in several insects and was recently confirmed in Drosophila. Segmentally represented pairs of cells homologous to the B_AG also have been identified in abdominal neuromeres of the hawkmoth Manduca sexta and other insects, in which the anatomy of their projections closely resembles that described in this study. The large amount of Bursicon expressed in the axons of these neurons after they leave the CNS supports the conclusion that they are responsible for most, if not all, of the hormone released into the blood. Once in the blood, the hormone has been shown not only to activate tanning, in part by upregulating epidermal tyrosine hydroxylase, but also to alter the physiology of the wing. Early evidence that Bursicon plasticizes the cuticle of the wing before expansion in Manduca has been confirmed recently using recombinant hormone, and genetic evidence from Drosophila indicates that Bursicon mediates apoptosis of the wing epidermis after expansion as a prerequisite for the fusion of the two cuticular panels. Anatomical and functional evidence thus supports a humoral role for Bursicon released from the B_AG, with these neurons mediating changes in the wing cuticle that support expansion. This conclusion is consistent with the projection pattern of these neurons described here, as well as with the observation that selective suppression of the B_AG (i.e., in c929-Gal4>3× UAS-EKO animals) blocks wing expansion, even though this manipulation leaves expansional behaviors intact. Presumably the inhibition of Bursicon release into the hemolymph in these animals prevents the changes in cuticle plasticity required to render the wings pliable (Peabody, 2008).

The data presented in this study demonstrate that the Drosophila B_SEG, and not the B_AG, are required for wing expansion behaviors. The broad projection pattern of these neurons in the CNS is consistent with their targeting multiple motor systems to activate both air swallowing and abdominal contraction, although their precise targets remain to be determined. Work from Manduca suggests that the circuitry underlying wing expansion may be similar in this insect. Wing expansion in Manduca is known to require intact descending connections from the subesophageal ganglion, and hawkmoths have B_SEG homologues, which localize to the labial neuromere of the subesophageal ganglion and send descending projections to all posterior ganglia. Further work will be required to determine the generality of this functional neuronal architecture in insects. Blowflies appear to represent an exception insofar as Bursicon has been reported absent in the subesophageal ganglion in these animals. Instead, Bursicon is synthesized by neurosecretory cells of the brain, which have been implicated in wing expansion behaviors. Reexamination of these conclusions, which predate the availability of antibodies to the hormone and were based on tanning bioactivity assays, should help clarify the extent to which Bursicon-expressing neurons in the subesophageal ganglion are likely to play a common role in insects (Peabody, 2008).

The widespread release of Bursicon in the nervous system, evidenced by the depletion of anti-Burs immunostaining from the B_SEG fibers after eclosion, suggests that centrally secreted Bursicon has multiple functions. The discovery that rickets mutants, which lack a functional Bursicon receptor, exhibit diminished humoral release of Bursicon from the B_AG points to a role beyond behavioral control. It remains to be shown that Bursicon acts as a centrally derived paracrine factor in potentiating its release from the B_AG, but there is reason to believe that the B_AG may be direct targets of Bursicon. Bursicon signaling is mediated by the cAMP pathway, and it has been shown that suppression of protein kinase A (PKA), one of the principal effectors of this pathway, decreases the release of Bursicon. Although this reduction was not sufficient to disrupt wing expansion and tanning, more recent experiments demonstrate that greater suppression of PKA reduces Bursicon release sufficiently to cause highly penetrant wing expansion deficits. This is consistent with the observation that overexpression of a cAMP phosphodiesterase (UAS-dunce) in the B_AG using c929-Gal4 results in wing expansion deficits in many flies when two copies of UAS-dunce are expressed (Peabody, 2008).

The conclusion that centrally secreted Bursicon participates in regulating its release as a hormone from the B_AG is interesting in light of the long-standing observation that Bursicon release into the hemolymph requires descending signals from the head. Decapitation or neck ligation of both blowflies and Drosophila soon after eclosion prevents tanning and wing expansion. Incisions that sever the ventral nerves in the neck of blowflies have also been reported to prevent tanning, but not air swallowing, suggesting that air swallowing, like tanning, is initiated by a signal that originates in the head. Although it is unclear that the circuitry governing Bursicon release is conserved in both types of fly, this observation is consistent with a mechanism in which Bursicon secreted by the B_SEG acts within the subesophageal ganglion or brain to initiate air ingestion and as a descending signal to promote Bursicon secretion from the B_AG. Since disruption of the Bursicon signaling pathway only partially attenuates Bursicon release from the B_AG, other regulatory signals must also exist (Peabody, 2008).

The discovery that the apoptosis of B_AG neurons is delayed in rickets mutants indicates that Bursicon promotes cell death in the CNS, as it does in the wing. Apoptosis of the B_AG and other CCAP-expressing neurons in the ventral ganglia is regulated by declining titers of 20-hydroxyecdysone (20E), which induces expression of the cell death genes reaper and hid. Bursicon presumably facilitates one or more steps in this process. Promotion of apoptosis may, in fact, be a general role of Bursicon in the CNS, given that many neurons in the thoracic and abdominal ganglia have been shown to die after eclosion in response to a head-derived signal, the release of which correlates closely with wing expansion. Further work will be required to test this hypothesis, but since the circuitry and musculature required for molting is substantially eliminated after eclosion, it would be parsimonious if Bursicon, which mediates the final physiological events of the terminal molt, were also to facilitate the demise of this machinery (Peabody, 2008).

In summary, these results provide key insights into the cellular mechanisms underlying Bursicon's regulation of wing expansion in Drosophila. Because wing expansion follows adult ecdysis, Bursicon's release is almost certainly modulated by the hormones that govern the events underlying that process. A remaining challenge is thus to understand the integrative mechanisms that coordinate the release of Bursicon with that of ecdysis-related hormones, such as ecdysis-triggering hormone and eclosion hormone. The functional architecture of the Bursicon system described here should inform these investigations and help elucidate the cellular circuitry that more generally mediates somatic and behavioral coordination during the ecdysis and postecdysis phases. These results have demonstrated that Bursicon contributes more globally to postecdysis than originally imagined (Peabody, 2008).

DEVELOPMENTAL BIOLOGY

The neurons that express the bursicon gene were identified using in situ hybridization. The bursicon antisense probe revealed one to two pairs of neurons in each of the thoracic and abdominal neuromeres of the larval CNS. Since Bursicon is co-expressed in some of the neurons that express the neuropeptide CCAP (see Drosophila CCAP) in a number of other insects (Honegger, 2002; Kostron, 1996), whether this relationship also held for Drosophila was examined. By double labeling with an antiserum directed against CCAP, it was found that bursicon transcripts are indeed expressed in a set of the neurons that contain CCAP. However, as in other insects (Honegger, 2002), not all CCAP neurons express bursicon. Bursicon transcripts were not detected in two pairs of CCAP-immunoreactive (ir) neurons in the brain or in two pairs in the first thoracic neuromere. Antibodies directed against amino acids 91-108 of the Bursicon sequence also labeled the CCAP-ir cells in the ventral nervous system (VNS). Using this antibody, a wild-type pattern of Bursicon-ir neurons was detected in the burs^Z5569 and burs^Z1091 mutants, indicating that these mutants produce Bursicon protein, although it has greatly reduced biological activity (Dewey, 2004).

An neuronal network responsible for the regulation and release of Bursicon

A subset of Drosophila neurons that expresses crustacean cardioactive peptide (CCAP) makes the hormone bursicon, which is required for cuticle tanning and wing expansion after eclosion. Evidence is presented that CCAP-expressing neurons (N_CCAP) consist of two functionally distinct groups, one of which releases bursicon into the hemolymph and the other of which regulates its release. The first group, which it called N_CCAP-c929, includes 14 bursicon-expressing neurons of the abdominal ganglion that lie within the expression pattern of the enhancer-trap line c929-Gal4. Suppression of activity within this group blocks bursicon release into the hemolymph together with tanning and wing expansion. The second group, which has been called N_CCAP-R, consists of N_CCAP neurons outside the c929-Gal4 pattern. Because suppression of synaptic transmission and protein kinase A (PKA) activity throughout N_CCAP, but not in N_CCAP-c929, also blocks tanning and wing expansion, it is concluded that neurotransmission and PKA are required in N_CCAP-R to regulate bursicon secretion from N_CCAP-c929. Enhancement of electrical activity in N_CCAP-R by expression of the bacterial sodium channel NaChBac also blocks tanning and wing expansion and leads to depletion of bursicon from central processes. NaChBac expression in N_CCAP-c929 is without effect, suggesting that the abdominal bursicon-secreting neurons are likely to be silent until stimulated to release the hormone. These results suggest that N_CCAP form an interacting neuronal network responsible for the regulation and release of bursicon and suggest a model in which PKA-mediated stimulation of inputs to normally quiescent bursicon-expressing neurons activates release of the hormone (Luan, 2006).

Tanning bioassays performed in blowflies and the hawkmoth, Manduca sexta, have determined that bursicon bioactivity is concentrated in the abdominal ganglia from which it is likely to be released into the hemolymph. Recent molecular characterization of bursicon, and the availability of antibodies to its two subunits, has allowed identification of neurons that make bursicon in several insects and confirmed previous findings that some of these coexpress CCAP, a peptide with cardioacceleratory activity. In Drosophila larvae, bursicon expression is restricted to a small number of CCAP-expressing neurons in the ventral nerve cord. Its distribution has now been mapped in late-stage pharate adults, a stage more relevant to its release into the hemolymph. Bursicon expression in the adult is broader but remains restricted to N_CCAP, with most bursicon-expressing neurons located in the abdominal ganglion. The 14 abdominal neurons (B_AG) are included in the expression pattern of the c929-Gal4 enhancer-trap line, whereas a pair of neurons that consistently express bursicon in the subesophageal ganglion (B_SEG) are not. Although bursicon immunoreactivity is occasionally observed in neurons other than these 16, the variability of its expression rendered these neurons unlikely substrates for the highly invariant developmental processes of cuticle tanning and wing expansion that bursicon mediates (Luan, 2006).

Functional evidence that the B_AG are responsible for release of bursicon into the hemolymph is provided by demonstrating that suppression of excitability in the c929-Gal4 pattern blocks bursicon release into the hemolymph. The inhibition of wing expansion by this manipulation suggests that wing expansion, like tanning, also requires bursicon in the hemolymph. This is consistent with a proposed role for bursicon in cuticle plasticization, a process required to render the wing extensible before expansion. The partially expanded wing phenotypes seen at lower levels of suppression may result from incomplete cuticle plasticization attributable to insufficient bursicon in the hemolymph (Luan, 2006).

The expression of c929-Gal4 in B_AG, but not B_SEG, may indicate functional distinctions between these two groups of neurons. The c929-Gal4 expression pattern has been extensively characterized and conforms primarily to that of dimmed, a gene that neighbors the Gal4 insertion site and is involved in upregulating peptide processing, and the coincidence of bursicon and c929-Gal4 expression in B_AG may reflect upregulation of peptidergic processing in N_CCAP neurons preparing to corelease both CCAP and bursicon into the hemolymph. B_SEG, which lie outside the c929-Gal4 expression pattern and are unlikely to contribute significantly to circulating levels of bursicon in the hemolymph after eclosion based on these Western blot data, may instead release the hormone within the CNS, where it may regulate behaviors required for wing expansion (Luan, 2006).

This study introduces a new tool for the targeted enhancement of cellular excitability; it was used to demonstrate that secretion of bursicon from B_AG must be regulated by a population of N_CCAP neurons outside of N_CCAP-c929 (i.e., by N_CCAP-R). Evidence is provided that B_AG are electrically quiescent before eclosion. The tool is a GFP-tagged version of the bacterial sodium channel NaChBac. Transgenic flies were made that express UAS-NaChBac-EGFP under the control of Gal4 drivers; it was shown that NaChBac-EGFP enhances photoreceptor excitability. (Nitabach, 2005), and the utility of the NaChBac channel in enhancing excitability was further demonstrated in other cell types (Luan, 2006).

This study shows that enhancement of excitability in N_CCAP using NaChBac-EGFP eliminates bursicon secretion into the hemolymph and blocks tanning and wing expansion. This observation is consistent with results (Hodge, 2005) that show that dominant-negative inhibition of Shaw K⁺ channel function in N_CCAP results in many animals exhibiting the juvenile phenotype, particularly females. Interestingly, a similar sexual dimorphism was observed in the effects of NaChBac in N_CCAP when using a 'weaker' insert that lacks the EGFP tag. The Shaw channel is expressed endogenously in N_CCAP, and its properties suggest that it acts to limit membrane excitability. Inhibiting Shaw should therefore enhance excitability like NaChBac-EGFP. Conversely, overexpressing Shaw should suppress excitability and, like EKO, cause tanning and wing expansion deficits (Luan, 2006).

The observation that bursicon secretion into the hemolymph is relatively unimpaired by expression of NaChBac-EGFP in N_CCAP-c929 implies that NaChBac-EGFP does not act by directly enhancing the excitability of B_AG. Instead, NaChBac-EGFP must alter B_AG activity when it is expressed in neurons in N_CCAP-R. The failure of NaChBac-EGFP to affect bursicon release when expressed by c929-Gal4 is unlikely to result from lower transgene expression levels in B_AG than are obtained with CCAP-Gal4, because both drivers cause similar levels of wing expansion failure when driving expression of varying copy numbers of the EKO transgene (Luan, 2006).

How enhancement of excitability in N_CCAP impairs bursicon release remains an open question. The observation that NaChBac-EGFP depletes bursicon immunoreactivity in central processes when expressed in N_CCAP-R suggests a constitutive enhancement of bursicon secretion. If the central processes derive from B_SEG, this effect may be a direct consequence of their enhanced excitability. Enhanced excitability in B_AG, by expression of NaChBac-EGFP in N_CCAP-c929, does not deplete central bursicon or alter secretion of the hormone into the hemolymph after eclosion. This observation implies that B_AG are quiescent until stimulated after eclosion. This conclusion supports electrophysiological data from Manduca, which indicates that bursicon-secreting neurons receive little synaptic input until after eclosion when synaptic activity becomes continuous (P. Taghert, personal communication to Luan, 2006). More work will be required to determine how NaChBac-EGFP expression in N_CCAP-R alters bursicon secretion into the hemolymph (Luan, 2006).

The current results strongly support a role for synaptic transmission in the regulation of bursicon secretion. Inhibition of both dynamin and synaptobrevin function, by expression of UAS-Shi^ts1 and UAS-TNT, respectively, had no effect on wing expansion when expressed in N_CCAP-c929 alone, indicating that bursicon secretion is not dependent on these molecules. The observation that both UAS-Shi^ts1 and TNT inhibit wing expansion when expressed throughout N_CCAP therefore demonstrates that synaptic blockade in N_CCAP-R, but not N_CCAP-c929, is necessary for bursicon release. Block of synaptic inputs onto bursicon-secreting neurons may mediate this effect, although the efficacy of blockade in inhibiting wing expansion when applied before, as well as after, eclosion suggests the involvement of multiple synapses (Luan, 2006).

The less penetrant effects of TNT on wing expansion compared with those of UAS-Shi^ts1 cannot currently be explained. Tetanus toxin may not completely eliminate synaptic transmission. Alternatively, compensatory developmental responses to constitutive, rather than transient, block of synaptic transmission may be attenuating the effects of TNT. Changes in cellular physiology in response to TNT expression in neurons have been described previously (Luan, 2006).

PKA activity is required within N_CCAP for bursicon release. Inhibition of PKA within N_CCAP-c929 blocks neither tanning nor wing expansion. PKA is thus required within N_CCAP-R. Although the targets of PKA in N_CCAP remain to be determined, one intriguing possibility is that PKA downregulates the Shaw channel, which has been proposed to be a PKA target in mushroom bodies, to increase excitability and signaling in N_CCAP-R (Luan, 2006).

Additional work will be required to validate the model of B_AG regulation and to determine the functional roles of specific neurons within N_CCAP-R. Related to this is the question of whether bursicon secretion from B_SEG and B_AG is coordinately or independently regulated. Interestingly, the B_SEG neurons belong to N_CCAP-R and may themselves participate in regulating B_AG function. The role of the four non-bursicon-expressing neurons within N_CCAP-c929 also requires additional investigation. The data rule out a role for these neurons in regulating B_AG by mechanisms sensitive to enhancement of excitability or suppression of PKA or neurotransmission, but it remains possible that they regulate B_AG by other means (Luan, 2006).

The functionally oriented approach established here has facilitated the demonstration that the molecularly related N_CCAP neurons comprise all or part of a neuronal network that regulates bursicon secretion. Using this approach with enhancer-trap lines other than c929-Gal4 should allow the functional identities of specific neurons within this network to be defined (Luan, 2006).

A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster

A neuropeptide hormone-signalling pathway controls events surrounding eclosion in Drosophila. Ecdysis-triggering hormone, eclosion hormone and crustacean cardioactive peptide (CCAP) together control pre-eclosion and eclosion events, whereas bursicon, through its receptor Rickets (RK), controls post-eclosion development. Cuticular tanning is a convenient visible marker of the temporally precise post-eclosion developmental progression, and this study investigated how it is controlled by the ecdysis neuropeptide cascade. Together, two enzymes, tyrosine hydroxylase (TH, encoded by ple) and dopa decarboxylase (DDC, encoded by Ddc), produce the dopamine that is required for tanning. Levels of both the ple and Ddc transcripts begin to accumulate before eclosion, coincident with the onset of pigmentation of the pharate adult bristles and epidermis. Since DDC activity is high before the post-eclosion onset of tanning, a different factor must be regulated to switch on tanning. Transcriptional control of ple does not regulate the onset of tanning because ple transcript levels remain unchanged from 24 hours before to 12 hours after eclosion. TH protein present before eclosion is degraded, and no TH activity can be detected at eclosion. However, TH protein rapidly accumulates within an hour of eclosion, and evidence is provided that CCAP controls this process. Furthermore, TH is shown to be transiently activated during tanning by phosphorylation at Ser32, as a result of bursicon signalling. It is concluded that the ecdysis hormone cascade acts as a regulatory switch to control the precise onset of tanning by both translational and activational control of TH (Davis, 2007).

In Drosophila, the onset of tanning of the puparium occurs within 1 hour after the wandering larva becomes sessile. This requires metabolites of DA, the production of which is dependent on the actions of TH and DDC. Transcripts levels of both genes, and TH protein and activity levels, are all high in white pre-pupae (WPP). Unlike at eclosion, TH does not appear to be activated by PKA phosphorylation for the rapid tanning of the pupal case at pupariation. This is not unexpected because the ecdysis neuropeptides are not released until a full 12 hours APF. During the late third instar, the relatively insoluble tyrosine, which is indispensable for tanning, is stored as a more soluble derivative, tyrosine-O-phosphate (tyr-P). Tanning at pupariation is probably controlled by the release of tyrosine from tyr-P. No appreciable accumulation of tyr-P occurs before eclosion, suggesting that post-eclosion tanning is switched on by a different mechanism (Davis, 2007).

This study establishes a role for the ecdysis neuropeptide cascade in post-eclosion tanning by examining the regulation of two genes, ple and Ddc, which encode two enzymes with critical roles in tanning. Semi-quantitative RT-PCR was used to examine the profile of transcription after puparium formation. Levels of both transcripts are high in WPP, but they drop and then rise again before eclosion. Ddc levels begin to increase 60 hours APF, reach their peak 84 hours APF, and decline thereafter. DDC enzyme activity is required before eclosion for pigmentation of the pharate adult bristles and epidermis and after eclosion for tanning of the adult cuticle, and reaches a peak at eclosion. This indicates that Ddc is transcribed and translated before eclosion to ensure enzyme activity is present when substrate becomes available. This study investigated whether the control of substrate availability, and therefore the control of tanning, is effected by the transcriptional, translational, or post-translational regulation of TH (Davis, 2007).

Levels of ple transcripts are high during the 24 hour period spanning eclosion. The early appearance of ple transcripts is not surprising, because pigmentation of the pharate adult bristles and epidermis occurs between 84 and 96 hours APF. Both ple (and Ddc) transcription are normal in EH-KO, CCAP-KO, burs^Z1091 and rk⁴ flies. The accumulation of ple transcripts before eclosion, the maintenance of high levels of TH transcription until 12 hours after eclosion and the fact that neuropeptide mutant and ablation knockout flies exhibit normal ple transcription, led to the conclusion that the precise onset of tanning following eclosion is not due to regulation of ple transcription (Davis, 2007).

TH protein and activity levels are high before eclosion when pigmentation of the pharate adult bristles and epidermis occurs. Levels fall rapidly just before eclosion and rise thereafter. During this entire time, ple transcripts are present, suggesting that protein levels are being regulated. The drop in TH protein levels may occur through repression of translation from ple transcripts and/or increased turnover of the protein. The complete failure of CCAP-KO flies to accumulate TH protein following eclosion, although they transcribe ple normally, indicates a role for CCAP in this process. This could occur at the level of translation; alternatively, CCAP signalling may alter TH protein stabilisation. Since PKA signalling has been shown to regulate proteins involved in translational control, it is more likely that CCAP signalling activates PKA to cause translation, not stabilisation, of TH following eclosion (Davis, 2007).

EH-KO, burs^Z1091 and rk⁴ flies all appear to have relatively normal TH protein and activity profiles. Although all three exhibit a considerable range of activity in WPP, the pupal cases of these organisms tan normally. Despite the initial delay in TH accumulation in EH-KO flies following eclosion, these flies, and burs^Z1091 and rk⁴ mutants, maintain high levels of TH until 144 hours APF, a time when TH is undetectable in control flies. This persistence of TH indicates a delay in the execution of the neuropeptide hormone cascade. Interestingly, rk⁴ flies also show a delay in degradation of TH following pupariation. Perhaps there is a requirement for RK signalling to trigger TH degradation following tanning of the puparium (Davis, 2007).

Neck-ligation of flies at eclosion prevents tanning, whereas flies ligated 30 minutes after eclosion tan normally. Furthermore, tanning of flies neck-ligated at eclosion is rescued by injection of 8-Br-cAMP. TH protein begins to accumulate 1 hour after eclosion in control flies. Phosphorylation of the protein by PKA at Ser32 leads to enzyme activity rising between 1.5 and 3 hours after eclosion. It is concluded that the translational and activational state of TH is responsible for controlling tanning following eclosion. TH protein accumulates, but is not phosphorylated in flies neck-ligated at eclosion resulting in reduced TH activity. Interrupting neuropeptide signalling after eclosion reveals that the element that controls TH translation is released before eclosion. The loss of TH accumulation in CCAP-KO flies and the restoration of TH accumulation upon injection of CCAP suggests that CCAP is responsible for inducing TH translation (Davis, 2007).

Flies neck-ligated 30 minutes after eclosion, translate and phosphorylate TH normally. By allowing neuropeptide signalling after eclosion, it has been demonstrated that a factor is released within 30 minutes of eclosion that causes phosphorylation and therefore activation of TH. The reduced phosphorylation of Ser32 in burs^Z1091 flies, and complete loss of phosphorylation in rk⁴, burs^Z1091/burs^Z5569 and rk¹/rk⁴ flies suggests that bursicon signalling through RK controls this process. Activity levels of TH are significantly reduced in flies neck-ligated at eclosion compared with control flies. Flies ligated 30 minutes after eclosion show twofold higher levels than flies neck-ligated at eclosion and this difference probably accounts for the presence or absence of tanning. This suggests that a critical threshold of TH activity exists that is surpassed in the flies ligated at 30 minutes. Thus, although the activity present in these flies is significantly less than that in control flies, the organisms have sufficient TH activity to tan, whereas flies ligated at eclosion do not attain the threshold of activity required for tanning. Injection of 8-Br-cAMP into flies neck-ligated at eclosion rescues tanning by restoring phosphorylation and therefore activation of TH. Although injection of 8-Br-cAMP does not restore TH activity to control levels, it increases activity nearly sixfold, achieving the threshold of activity required for tanning following eclosion (Davis, 2007).

These results, taken together, suggest that at least two factors control the precise timing of tanning after eclosion. One, released before eclosion, causes translation of TH; the other, released after eclosion, causes phosphorylation and activation of TH. Both EH and CCAP are released before eclosion to control pre-ecdysis and ecdysis, respectively. EH-KO and CCAP-KO flies both exhibit extreme post-eclosion tanning defects. EH-KO flies take more than 9 hours to tan and CCAP-KO flies fail to tan. TH protein is undetectable in EH-KO flies immediately following eclosion, but these flies do eventually accumulate TH and tan. The complete failure of CCAP-KO flies to tan, combined with the fact that CCAP-KO flies fail to accumulate TH from the ple transcripts that are present at eclosion, suggest that CCAP is responsible for inducing TH translation. The initial failure of EH-KO flies to accumulate TH is probably caused by a failure to trigger the rapid release of CCAP. Presumably, enough CCAP is eventually released in these flies to effect the translation of TH and eventually tanning, because the EH genetic ablation is leaky. Consistent with this prediction, EH-KO flies that expand their wings accumulate TH normally, suggesting that CCAP is released normally in these flies. TH translation is restored in CCAP-KO flies injected with CCAP and rescue of TH accumulation and phosphorylation occurs when EH-KO and CCAP-KO flies are injected with 8-Br-cAMP. Rescue of both defects probably occurs because injection of 8-Br-cAMP activates PKA in CCAP target cells, thus circumventing the need for CCAP release, and also activates PKA in TH-expressing cells, leading to phosphorylation and activation of TH (Davis, 2007).

These data suggest that the post-eclosion factor causing the phosphorylation of Ser32 is the heterodimeric hormone bursicon. It is responsible for tanning and wing expansion and acts through its receptor RK. Consistent with the role of bursicon in the phosphorylation of TH, rk⁴ flies fail to phosphorylate TH and have reduced activity. These flies show a delay in tanning, taking up to 9 hours to tan. Injection of 8-Br-cAMP rescues tanning by restoring phosphorylation and therefore activation of TH (Davis, 2007).

Two mutants in the α subunit of bursicon have been identified, of which one (burs^Z5569) shows a delay in tanning in 40% of the progeny, whereas a delay is present in 82% of burs^Z1091/burs^Z5569 flies. The burs^Z1091 mutant does not show a delay in tanning, although phosphorylation of TH is reduced in these flies. Phosphorylation of Ser32 is undetectable in burs^Z1091/burs^Z5569 flies, probably causing the more severe tanning defect seen in these flies. The reduced phosphorylation of TH in burs^Z1091 flies corresponds to a minor loss of TH activity. Thus, it seems that the threshold TH activity required for proper tanning is achieved in burs^Z1091 flies, although they do not have wild-type levels of TH phosphorylation or activity. Normal tanning in these flies cannot be attributed to residual activity of the β subunit of bursicon, CG15284, proposed to be encoded by pu (S. McNabb and J. Truman, personal communication to Davis, 2007), because neither subunit independently confers bursicon activity. The burs^Z1091 allele is probably a hypomorph, and creation of a null allele would be useful. Additional studies on the activational state of TH in pu or burs null mutants will help to elucidate why tanning is not delayed in burs^Z1091 flies (Davis, 2007).

The data indicate that CCAP is responsible for initiating TH translation following eclosion. In Drosophila, translational regulation often occurs through microRNA (miRNA)-dependent RNAi-mediated repression through binding sites in the 3'UTR of transcripts. Three miRNAs - let-7, mir-iab-4-3p and mir-iab-4-5p - have been predicted to regulate TH translation in Drosophila. It is conceivable that one or more of these miRNAs, in association with the RISC complex, could bind to ple transcripts to cause the repression of translation through a miRNA-dependent RNAi-mediated mechanism. It is also plausible that PKA, activated by CCAP signalling, might relieve repression of TH translation by phosphorylation of one of the subunits of the RISC complex or associated proteins. Future work will establish whether there is a role for these miRNAs in the repression of TH translation before eclosion (Davis, 2007).

Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel

After emergence, adult flies and other insects select a suitable perch and expand their wings. Wing expansion is governed by the hormone Bursicon and can be delayed under adverse environmental conditions. How environmental factors delay Bursicon release and alter perch selection and expansion behaviors has not been investigated in detail. This study provides evidence that in Drosophila the motor programs underlying perch selection and wing expansion have different environmental dependencies. Using physical manipulations, it was demonstrated that the decision to perch is based primarily on environmental valuations and is incrementally delayed under conditions of increasing perturbation and confinement. In contrast, the all-or-none motor patterns underlying wing expansion are relatively invariant in length regardless of environmental conditions. Using a novel technique for targeted activation of neurons, this study shows that the highly stereotyped wing expansion motor patterns can be initiated by stimulation of N_CCAP, a small network of central neurons that regulates the release of Bursicon. Activation of this network using the cold-sensitive rat TRPM8 channel is sufficient to trigger all essential behavioral and somatic processes required for wing expansion. The delay of wing expansion under adverse circumstances thus couples an environmentally sensitive decision network to a command-like network that initiates a fixed action pattern. Because N_CCAP mediates environmentally insensitive ecdysis-related behaviors in Drosophila development before adult emergence, the study of wing expansion promises insights not only into how networks mediate behavioral choices, but also into how decision networks develop (Peabody, 2009).

The introduction of genetically encoded effectors, which permit the targeted manipulation of neuronal activity in vivo, is increasingly facilitating neurobiological studies of behavioral choice in vivo, as exemplified by the work presented in this study. Using a new method for stimulating neurons in live animals, it was demonstrated that the N_CCAP network, first implicated in governing ecdysis, effects the fly's decision to expand its wings after eclosion. This decision is normally coupled to the decision to perch, which this study demonstrates to be based on evaluation of environmental variables. The postponement of wing expansion by the fly under adverse circumstances is thus a consequence of a value-based choice to prolong search behavior and the delayed activation of a command network responsible for the execution of the wing expansion decision (Peabody, 2009).

The results support a model in which the motor programs underlying perch selection respond primarily to environmental input. Although the environmental features monitored by the fly and the sensory channels that process them remain to be determined, it is clear from the experiments that flies assess conditions during phase I and assign longer search periods to more adverse environments. The observation that expansion is not deferred indefinitely, even under the high-perturbation condition, suggests that the benefits of continued searching are weighed against the risks of further delay. How these risks, which may include predation and desiccation, are represented physiologically to encode the 'value' of different environments is still unclear, but the work described in this study should facilitate their investigation (Peabody, 2009).

Elucidating which neurons mediate the decision to perch will also require further investigation. Although perching can be induced by the activation of N_CCAP using UAS-TRPM8, N_CCAP is clearly not normally required for terminating phase I, since its suppression does not affect perching. Also, the perching that does occur when N_CCAP is stimulated by UAS-TRPM8 is more tightly linked temporally with the initiation of expansion (i.e., phase III) than with the onset of N_CCAP stimulation (i.e., the shift to 18°C), suggesting that neurons downstream of N_CCAP mediate phase I termination, most likely at the level of the motor networks underlying perch selection and expansion. Inhibition of one motor system by another has been demonstrated to explain such behavioral hierarchies in other cases, as in the dominance of feeding over withdrawal in mollusks (Peabody, 2009).

In contrast to perch selection, the expansion program shows no obvious environmental dependence, but it is strongly dependent on levels of N_CCAP activity for its initiation. This is consistent with the known effects of Bursicon, which is released by a subset of N_CCAP and is required for both wing extensibility and phase III behaviors. The probability of expansion occurring at all is likely to depend on whether the Bursicon released into the CNS reaches a critical threshold, while the initiation of expansion most likely depends on the timing and/or rate of Bursicon release. The rate of release should decline with N_CCAP suppression, which will reduce the excitability of the Bursicon-expressing neurons, and may account for both the prolongation of phase II under this condition and the appearance of graded wing expansion deficits. It is also possible that the timing of Bursicon release was modulated by manipulations of N_CCAP if delay circuits intrinsic to N_CCAP are responsible for initiating its secretion (Peabody, 2009).

Although it remains to be determined whether N_CCAP participates in the decision to expand, it is clear that its activation drives all the behavioral and somatic processes necessary for wing expansion, placing N_CCAP neurons high in the execution pathway of the decision and indicating that their function in wing expansion is command-like. Because this command-like function relies on Bursicon and not direct synaptic activation, it differs from familiar command systems that mediate fast behavioral switches, such as those involved in defensive escape, and more closely resembles systems in which hormones or neuromodulators elicit profound behavioral transitions such as those responsible for egg-laying in mollusks, stomatogastric ganglion modulation in crustacea, and ecdysis in insects. Because of the technical difficulty involved, the ability of such systems to drive behavioral programs upon activation has rarely been demonstrated. However, as illustrated by the work presented in this study, the introduction of genetic techniques for in vivo neuronal activation should make such demonstrations increasingly possible (Peabody, 2009).

The work presented in this study argues that the motor programs for perch selection and wing expansion have distinct regulatory mechanisms, but leaves unanswered the question of how the decisions to perch and expand are coupled: Specifically, the mechanism by which N_CCAP is activated following perching remains to be clarified. N_CCAP could, in principle, receive input from either the perch selection motor network or from sensory processing pathways. Such input is likely to be indirect since perching and expansion are separated by a delay (i.e., phase II), which varies considerably in duration between individual animals and different conditions. This delay period, during which Bursicon secretion is initiated, may act, at least in part, to permit Bursicon to plasticize the cuticle of the wing and body before expansion (Peabody, 2009).

Behavioral transitions during ecdysis in both Drosophila and the hawkmoth Manduca sexta, are known to be regulated by inhibitory delay circuits. In the case of eclosion hormone, the delay circuit temporally separates the somatic and behavioral aspects of the hormone's action and has been proposed to act as a control point for environmental modulation by light, which accelerates eclosion, perhaps by suppressing the inhibitory delay circuit. Interestingly, the modulation of eclosion by light is eliminated in animals lacking N_CCAP, supporting a role for these neurons in the inhibitory pathway. Because ablation of the EH-expressing neurons causes wing expansion deficits, it is possible that in addition to modulating eclosion, EH may similarly modulate wing expansion by simultaneously upregulating excitability in both the Bursicon-expressing neurons and in a delay circuit that inhibits them. By analogy to the action of EH on ecdysis, the latter circuit, which could include non-Bursicon-expressing N_CCAP neurons, would inhibit Bursicon release until inhibition is alleviated by an environmentally mediated signal. Such a model might explain not only the wing expansion deficits observed in animals lacking EH-expressing neurons, but also the eventual wing expansion of animals kept in the high-perturbation condition. In these animals, run down in the delay circuit may trigger Bursicon release before the decision to perch, as happens when N_CCAP is activated by UAS-TRPM8, and thus also account for their abbreviated phase II (Peabody, 2009).

The merits of this model, and others that invoke delay circuits, remain to be tested. It is interesting to note, however, that the mechanism(s) coupling environmental input to N_CCAP is likely to be added during metamorphosis, because the function of this network changes from an intrinsically regulated mediator of pupal ecdysis to an extrinsically modulated mediator of wing expansion. Despite its broad phylogenetic conservation, this network also appears to be differentially regulated in different species, with CCAP-expressing neurons being direct targets of EH in many insects, but not Drosophila. Such differential regulation may underlie, at least in part, species-specific differences in the coordination of wing expansion with adult emergence: In some insects, expansion is initiated at emergence without benefit of environmental input, and in others, like honeybees, it is initiated and completed before emergence. Investigation of the basis of these differences, and of the neural mechanisms that adapt N_CCAP function to the individual developmental needs and life histories of different insects, should provide insight into general mechanisms of behavioral adaptation (Peabody, 2009).

In general, the work presented in this study demonstrates that the behavioral programs used by Drosophila to achieve wing expansion can serve as a simple and fruitful model for investigating networks underlying behavioral choice. Elucidation of the neuronal pathways that mediate the decisions to perch and expand the wings will shed light on the development and evolution of behavioral networks as well as their architecture and function. Indeed, the decision-making architecture outlined here for wing expansion may prove relevant to the type of instinctive behaviors in which an environmentally sensitive 'appetitive' phase is coupled to a 'consummatory' phase consisting of a stereotyped, motor pattern. Finally, tools that permit one to manipulate decision-making in living, behaving animals, such as UAS-TRPM8, which is introduced in this study, clearly will play an essential role in mapping the neural networks underlying behavior in Drosophila and other animals (Peabody, 2009).

Chip is required for posteclosion behavior in Drosophila

Neurons acquire their molecular, neurochemical, and connectional features during development as a result of complex regulatory mechanisms. This study shows that a ubiquitous, multifunctional protein cofactor, Chip, plays a critical role in a set of neurons in Drosophila that control the well described posteclosion behavior. Newly eclosed flies normally expand their wings and display tanning and hardening of their cuticle. Using multiple approaches to interfere with Chip function, it was found that these processes do not occur without normal activity of this protein. Furthermore, the nature of the deficit was identified to be an absence of Bursicon in the hemolymph of newly eclosed flies, whereas the responsivity to Bursicon in these flies remains normal. Chip interacts with transcription factors of the LIM-HD (LIM-homeodomain) family, and one member, dIslet, was identified as a potential partner of Chip in this process. These findings provide the first evidence of transcriptional mechanisms involved in the development of the neuronal circuit that regulates posteclosion behavior in Drosophila (Hari, 2008).

This study used a selective overexpression strategy to identify a novel function of Chip in a set of neurons that control a stereotyped behavioral program. Chip is a widely expressed multidomain cofactor molecule that interacts with many transcription factors. It can function both as a transcriptional coactivator and a bridging factor between proteins that bind to distal enhancers and the core transcription machinery. Identifying specific functions of such a protein is confounded by the superposition of a multitude of effects. The mutations in Chip cause early lethality precluding the examination of later functions. Because the molecule exists as a part of multiple complexes, even simultaneously within the same cell, altering the level of one class of interactors can potentially disrupt several functions. This study has elucidated a highly specific role of Chip in a particular class of neurons in Drosophila and implicated a known LIM-HD partner of Chip, Islet, in this function (Hari, 2008).

It is proposed that the defect is attributable to a failure in the release rather than in the production or the responsiveness to the neurohormone Bursicon. How might Bursicon release be controlled as a result of Chip function in development? The hemolymph transfer experiments provide a unique insight into this puzzle. The literature describes a model wherein posteclosion wing expansion requires a combination of a neural signal from the brain as well as Bursicon release. The results extend the understanding of how this interplay of activity and secreted factors is set up in development. It appears that, several days before eclosion, Chip is able to regulate an as-yet-unidentified event in the CCAP neurons, such that the hemolymph contains adequate levels of Bursicon after eclosion. The CCAP-expressing neurons are divided into at least two interacting subpopulations, only one of which secretes Bursicon. The other subpopulation does not secrete Bursicon but is implicated in regulating its release. Chip may therefore mediate the formation of proper connectivity among CCAP neurons, which eventually ensures timely Bursicon release several days later. Supporting this scenario, Chip has been reported to regulate axon pathfinding and proper innervation of targets in other systems. The data are suggestive of Chip requirement in the early period of puparium formation, which fits well with a report that CCAP neurons undergo extensive remodeling in this period of metamorphosis. The importance of this connectivity is underscored by the identification of several other genes in a gain-of-function screen, which displayed a simultaneous disruption of both posteclosion wing expansion and the pattern of CCAP neuron innervation. Therefore, thes findings motivate an examination of Chip function in regulating the connectivity of CCAP neurons, a role that directly links this key aspect of neuronal development with the control of posteclosion behavior in Drosophila (Hari, 2008).

Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. This study found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. This study establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, these data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates (Veverytsa, 2011).

Retrograde BMP signaling is required to maintain the behavioral output of neuronal networks. Collectively, these data show that retrograde BMP signaling upregulates the expression of a combination of peptide hormones, exclusively in the CCAP-EN subset of CCAP neurons and to a level required for those neurons to contribute to the normal execution of ecdysis behaviors. These findings in relation to the function of CCAP-ENs in ecdysis, as well as the utility of retrograde signaling as a conserved mechanism for differentiating neuronal identity and regulating behavior (Veverytsa, 2011).

A feed-forward peptide hormone cascade coordinates ecdysis. Larval and pupal pre-ecdysis is initiated by Ecdysis triggering hormone (ETH) from peripheral Inka cells stimulating Eclosion hormone (EH) secretion from brain Vm neurons. ETH and EH then act together on CCAP neurons to stimulate CCAP and Mip release. Work on the isolated Manduca central nervous system demonstrates that CCAP and MIP synergistically terminate pre-ecdysis and initiate ecdysis proper motor rhythm. This is supported by Drosophila studies; CCAP neuron ablation prolongs pre-ecdysis and ecdysis proper in larvae, and results in a deficit in the execution of the ecdysis program in pupae that reduces head and appendage eversion and extension. This role for CCAP neurons has largely been attributed to abdominal CCAP-INs acting locally on motoneurons. However, these observations indicate an essential role for BMP-dependent peptide hormone expression in CCAP-ENs. A detailed analysis of ETH-driven neuronal activity during Drosophila pupal ecdysis supports these conclusions. This study shows that T3 and A8/A9 CCAP neurons are active at the start of ecdysis proper, coincident with head eversion, and that A1-A4 CCAP neurons are active secondarily and throughout the remainder of ecdysis proper, coincident with appendage and head extension. It is suggested that the A1-A4 CCAP neurons active during pupal ecdysis proper and required for leg extension are CCAP-ENs. How would CCAP-ENs that secrete hormones into the hemolymph regulate ecdysis? It has been argued that hemolymph-borne CCAP, Mip and bursicon regulate heart rate, hemolymph pressure and cuticle expansion. However, these peptide hormones might also regulate the activity of central circuits, either indirectly or directly, as established for ETH. Genetic analysis of CCAP, Mip and bursicon peptide hormones and their receptors would provide valuable answers to these questions (Veverytsa, 2011).

CCAP-ENs require peripherally derived Gbb for BMP signaling and enhanced peptide hormone expression. CCAP-EN axons terminate on muscle 12. Muscle expresses Gbb and this study found that muscle-derived (but not neuronal-derived) Gbb significantly rescued BMP signaling and peptide hormone expression in CCAP-ENs. pMad immunoreactivity and GFP-Tkv (expressed from Ccap-GAL4) were also observed within type III boutons, indicative of local BMP signaling. Thus, together with reports that muscle-derived Gbb is sufficient for retrograde BMP signaling in motoneurons, the weight of evidence supports the somatic muscle as a primary target for Gbb access for CCAP-ENs. However, the possibility cannot be ruled out that other sources for Gbb exist, perhaps secreting the ligand into the circulating hemolymph. In this regard, it has been reported that, in gbb mutants, restoration of Gbb in another peripheral tissue, the fat body, failed to rescue BMP signaling in neurons, suggesting that distant signaling via the hemolymph is not sufficient. Further detailed analysis will be required to identify necessary and/or redundant roles for other tissues in neuronal BMP signaling (Veverytsa, 2011).

Although muscle is the likeliest target with respect to gbb, the muscle is unlikely to be the primary target for CCAP-EN peptide hormones. Ultrastructural analysis shows that type III boutons lie superficially on the muscle surface and that dense core vesicles exocytose towards the hemolymph and muscle. Furthermore, bursicon immunoreactivity is detectable in the hemolymph. CCAP-EN peptide hormones are known to target the wing, cuticle and cardiac and visceral muscle, but not the somatic muscle. This situation is unusual, as target-derived factors are typically viewed as influencing neuronal gene expression profiles pertinent to the target itself. Footpad-derived cytokines induce cholinergic differentiation of sympathetic neurons required for footpad sweat secretion. Axial differences in BMP4 ligand expression in the murine face direct subset-specific gene expression in innervating trigeminal neurons that shapes the formation of somatosensory maps. Activin and nerve growth factor in the developing skin induce expression of the hyperalgesic neuropeptide calcitonin gene-related peptide (CGRP) in nociceptive afferents (Veverytsa, 2011).

Without evidence for such a mutualistic relationship, what purpose could retrograde BMP-dependent gene expression play in CCAP-ENs? The tremendous cellular diversity of the nervous system is achieved through the progressive refinement of transcriptional cascades within increasingly diversified neuronal progenitor populations . Subsequently, retrograde signaling further differentiates the expression profile in postmitotic neurons. In such cases, unique access to extrinsic ligands allows for a certain mechanistic economy, enabling a somewhat common regulatory landscape to be adapted towards distinct gene expression profiles. In this context, it is postulated that retrograde BMP signaling functions to diversify the expression levels of peptide hormones in CCAP neurons. Drosophila interneurons and efferents can be sharply distinguished on the basis of BMP activity. Moreover, this study shows that BMP activation in CCAP-INs is capable of enhancing their peptide hormone expression, implicating a similar gene regulatory landscape in CCAP-ENs and CCAP-INs. Thus, the BMP dependence of CCAP, Mip and Bursß offers a simple solution to the problem of how to selectively enhance peptide hormone expression in CCAP-ENs (Veverytsa, 2011).

BMP signaling offers an additional advantage to neuronal diversification. Studies of axial patterning in Drosophila have unveiled a wealth of mechanisms that diversify and gauge transcriptional responses to BMP signaling. These mechanisms revolve around the outcome of pMad/Medea activity at a gene's cis-regulatory sequence, as influenced by their affinity for specific cis-regulatory sequences and local interactions with other transcription factors, co-activators and co-repressors. As a result, pMad/Medea activity can be extensively shaped to generate gene- and cell-specific responses and determine whether genes are on or off or up- or downregulated. This flexibility is likely to underpin the differential sensitivity of CCAP, Mip and Bursß to a common retrograde BMP signal within a single cell, as well as the utility of BMP signaling as a common retrograde regulator of subset-specific gene expression in distinct neuronal populations (Veverytsa, 2011).

Finally, the differential regulation of Bursα and Bursβ is intriguing because they are believed to only function as a heterodimer. Although the possibility of functional homodimers cannot be discounted, it is postulated that the selective BMP dependence of Bursβ might be an efficient mechanism for modulating the activity of the active bursicon hormone. This would be analogous, and perhaps orthologous, to the regulation of follicle-stimulating hormone in mammals. Its cyclical upregulation during the oestrous cycle is dictated by the regulation of only one of its subunits, FSHβ, by the TGFβ family ligand activin (Veverytsa, 2011).

Numerous studies have described the impact of retrograde signaling on neuronal network formation and function. During spinal sensory motor circuit development, retrograde neurotrophin signaling induces specific transcription factor expression in motoneurons and Ia afferents that is required for appropriate motor sensory central connectivity, which, when inoperative, results in ataxic limb movement. Similarly, murine trigeminal neurons utilize spatially patterned BMP4 expression in the developing face to target their centrally projecting axons in a somatotopically appropriate manner. Retrograde signaling also modulates physiologically responsive neuronal gene expression. In vertebrates, skin injury induces cutaneous activin and nerve growth factor expression, which retrogradely upregulates sensory neuron expression of CGRP, which mediates hyperalgesia. In sensory motor circuits of Aplysia, retrograde signals are required to upregulate presynaptic sensorin, a neuropeptide required for long-term facilitation of the sensorimotor synapse (Veverytsa, 2011).

The current evidence suggests that the function of BMP signaling is not mediated within a specific developmental window, but is required on an ongoing basis. The Ccap-GAL4 transgene is not active until late larval stage L1, after CCAP neuron network assembly and peptide hormone initiation. Yet, wit phenotypes were significantly rescued using Ccap-GAL4. Together with observation of persistent pMad immunoreactivity in CCAP-ENs, it is concluded that BMP signaling acts permissively to maintain the capacity of CCAP-ENs to contribute to ecdysis, rather than acting phasically at ecdysis to instructively activate ecdysis behaviors or enable CCAP-ENs to contribute. Such a maintenance role is supported by previous work showing that maintained expression of the neuropeptide FMRFa requires persistent retrograde BMP signaling. It was also found that type III synapses on muscle 12 have significantly fewer boutons and shorter branches in wit mutants, implicating a role for BMP signaling in CCAP-EN synaptic morphology, as first described for type I neuromuscular junctions in wit mutants. It will be of interest to investigate whether dense core vesicle exocytosis is also perturbed in wit mutants, akin to the reduced synaptic vesicle exocytosis at type I boutons in wit mutants (Veverytsa, 2011).

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).

Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

Although the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. This study shows in the fruit fly that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocks metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppresses the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulate overgrowth. Interestingly, InR displays little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation (Gu, 2014).

It is well established that insulin/insulin-like signaling (IIS) is crucial for regulating cell growth and division in response to nutritional conditions in Drosophila. However, most studies have focused on growth of the body or individual organs, and comparatively little is known about the roles of IIS during neuronal development, particularly in later developmental stages. Drosophila InR transcripts are ubiquitously expressed throughout embryogenesis, but are concentrated in the nervous system after mid-embryogenesis and remain at high levels there through the adult stage. This suggests that IIS plays important roles in the post-embryonic nervous system. Recently, analysis of Drosophila motorneurons, mushroom body neurons, and IPCs has revealed important roles of PI3K and Rheb in synapse growth or axon branching. These studies revealed some growth regulatory functions of IIS in the CNS, but they have not explored whether the control of neuronal growth by IIS is temporally regulated (Gu, 2014).

This study has shown that IIS strongly stimulates organizational growth of neurons during metamorphosis, whereas the effects of IIS on larval neurons are comparatively modest. Recently, similar results have been reported in mushroom body neurons, in which the TOR pathway strongly promoted axon outgrowth of γ-neurons after metamorphic pruning. Expression of FOXO or reduction of InR function had no significant effect on larval growth of the CCAP/bursicon neurons, or on the soma size of many other larval neurons. Thus, while IIS has been shown to regulate motorneuron synapse expansion in larvae, the current findings indicate that IIS may not play a major role in regulating structural growth in many larval neurons. This is consistent with a recent report that concluded that the Drosophila larval CNS is insensitive to changes in IIS (Gu, 2014).

When InR^act was used to activate IIS without ligand, a modest but significant increase was seen in the soma size of the more anterior CCAP/bursicon neurons during larval development. This result indicates that the IIS pathway is present and functional in these larval neurons, but the ligand for InR is either absent or inactive. During metamorphosis, unlike in larvae, downregulation of IIS by altering the level of InR or downstream components of the pathway significantly reduced CCAP/bursicon neuron growth. Thus, the results suggest that IIS is strongly upregulated during metamorphosis to support post-embryonic, organizational growth of diverse peptidergic neurons, and this activation may at least in part be due to the presence of as yet unidentified InR ligands during metamorphosis (Gu, 2014).

Attempts were to identify this proposed InR ligand source by eliminating, in turn, most of the known sources of systemic DILPs. None of these manipulations had any effect on metamorphic growth of the CCAP/bursicon neurons. These results are consistent with three possible mechanisms. First, there may be a compensatory IIS response to loss of some dilp genes. For example, a compensatory increase in fat body DILP expression has been observed in response to ablation of brain dilp genes. Second, the growth may be regulated by another systemic hormone (e.g. DILP8) that was not tested, or by residual DILP peptides in the RNAi knockdown animals. Third, a local insulin source may be responsible for stimulating metamorphic outgrowth of the CCAP/bursicon neurons. Consistent with this view, a recent report showed that DILPs secreted from glial cells were sufficient to reactivate neuroblasts during nutrient restriction without affecting body growth, while overexpression of seven dilp genes (dilp1-7) in the IPCs had no effect on neuroblast reactivation under the same conditions. It seems likely that glia or other local DILP sources play an important role in regulating metamorphic neuron growth, but further experiments will be needed to test this model (Gu, 2014).

When IIS was manipulated in the CCAP/bursicon neurons, changes were observed in cell body size (and sometimes shape) and in the extent of branching in the peripheral axon arbor. Although this study focused analysis of neurite growth on the peripheral axons, which are easily resolved in fillet preparations, corresponding changes were also observed in the size and complexity of the central CCAP/bursicon neuron arbor. These IIS manipulations (both upregulation or downregulation) resulted in the above structural changes as well as wing expansion defects, suggesting that the normal connectivity of the CCAP/bursicon neurons was required for proper functioning of this cellular network. This model is consistent with the observation of two subsets of morphologically distinct bursicon-expressing neurons (the B_SEG and B_AG neurons), which are activated sequentially to control central and peripheral aspects of wing expansion. The B_SEG neurons project widely within the CNS to trigger wing expansion behavior as well as secretion of bursicon by the B_AG neurons. In turn, the B_AG neurons send axons into the periphery to secrete bursicon into the hemolymph to control the process of tanning in the external cuticle. Therefore, manipulation of IIS within these neurons, and the changes in morphology that result, may disrupt the wiring and function of this network. However, because the possibility cannot be ruled out that these IIS manipulations also altered neuronal excitability, synaptic transmission, or neuropeptide secretion, this study relied on measurements of cellular properties (and not wing expansion rates) when assessing the relative effects of different IIS manipulations on cell growth (Gu, 2014).

The results indicate that IIS is critical for organizational growth, which occurs during insect metamorphosis but is also seen during neuronal regeneration in other systems. However, the regenerative ability of many neurons is age-dependent and context-dependent; immature neurons possess a more robust regenerative capacity, while the regenerative potential of many mature neurons is largely reduced. In particular, the adult vertebrate CNS displays very limited regeneration, in marked contrast to the regeneration abilities displayed by the peripheral nervous system. Recent studies in mice suggest that age-dependent inactivation of mTOR contributes to the reduced regenerative capacity of adult corticospinal neurons, and activation of mTOR activity through PTEN deletion promoted robust growth of corticospinal tract axons in injured adult mice. The current genetic experiments demonstrate a requirement for activity of TOR, as well as several other IIS pathway components both upstream and downstream of TOR, in controlling organizational growth of many peptidergic neurons. This suggests that under certain conditions, the activation of IIS may be a crucial component of the conversion of mature neurons to a more embryonic-like state, in which reorganizational growth either after injury or as a function of developmental stage is possible. Given the strong evolutionary conservation of these systems and the powerful genetic tools available to identify novel regulatory interactions in Drosophila, studies on the control of organizational growth in this species hold great promise for revealing factors that are crucial for CNS regeneration (Gu, 2014).

Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut

Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. This study reports a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts (beyond its known roles in development) as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2 (Rickets), the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. This study has therefore identified a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells (Scopelliti, 2014).

Bursicon, also known as the tanning hormone, has been studied for decades due its essential role as the last hormone in the cascade of Ecdysis. In all invertebrate metazoa, this endocrine cascade is fundamental to coordinate molting events during animal lifetime, and in holometabolous insects, such as Drosophila, it control metamorphosis. Fly gene expression data suggest that the endocrine hormones and their cognate receptors involved in key stages of development may have other roles during adult animal life. However, these functional roles are largely unknown. This study is the first to demonstrate a role of Bursicon beyond development (Scopelliti, 2014).

A model is suggested in which Bursicon from enteroendocrine cells in the posterior midgut acts through DLGR2 to increase the production of cAMP within the VM, a mesenchymal ISC niche. This signaling limits the production of niche-derived, EGF-like Vein, leading to ISC quiescence. Burs protein expression was detected via immunolabeling in approximately 50% of the enteroendocrine cells of the posterior midgut, which appeared in stochastic spatial distribution within the most posterior segment of the adult midgut. Given that the percentage of enteroendocrine cells expressing Burs remained constant, it is likely that Burs expression might label a subtype of enteroendocrine cells within the midgut (Scopelliti, 2014).

The evidence indicates that burs mRNA levels are upregulated in the midgut during the phase of relative ISC quiescence in mature animals under homeostatic conditions. Conversely, during the phase of growth of the young immature gut or the dysplastic phase of the aging gut (both characterized by relative high rates of ISC proliferation) burs levels were relatively low, and burs overexpression was sufficient to suppress ISC proliferation. Therefore, the results provide the first demonstration of a tissue-intrinsic role of enteroendocrine cells, which drives homeostatic stem cell quiescence in the adult Drosophila midgut. Future studies should further characterize the upstream mechanisms controlling Burs production in the midgut, which might be linked to the yet undefined events involved in the regulation of overall tissue size and proliferation (Scopelliti, 2014).

Enteroendocrine cells are well known for their ability to mediate interorgan communication via hormone secretion into the bloodstream. The results demonstrate a novel, local role for enteroendocrine cells as paracrine regulators of stem cell proliferation. Such a mechanism could be phylogenetically conserved and take place in the mammalian intestine and other tissues of the gastrointestinal tract. This may therefore represent an unappreciated but yet important function of these cells beyond their conventional endocrine role (Scopelliti, 2014).

Mammalian LGRs are thought to drive ISC proliferation acting as receptors for the Wnt agonists R-spondins, which are unrelated to Bursicon and absent in the fly genome. Accordingly, no changes were detected in either Wg levels or signaling in burs or rk mutant midguts (Scopelliti, 2014).

Unexpectedly for a Wnt agonists and positive regulators of ISC proliferation, recent studies suggest that LGRs can act as tumor suppressors in colorectal cancer. Moreover, mammalian LGRs have also been shown to be activated by alternative ligands and promote cAMP signaling after the binding of yet unknown ligand(s). Therefore, it is likely that an unidentified functional homolog of Bursicon may act as an additional LGR ligand in mammals, driving ISC quiescence by regulating mitogenic signals from the surrounding niche as described here. Remarkably, DLGR2 shows closer sequence homology to the still poorly characterized LGR4, which (consistent with the Drosophila data) is expressed by the murine intestinal smooth-muscle layers and can signal via cAMP production. Consistent with the model, a recent study correlates loss-of-function mutations in LGR4 with multiple types of human epithelial carcinomas. Therefore, the results uncovered a novel biological role for LGRs, which is likely to impact mammalian stem cell research by providing a mechanistic framework for the so far correlative mammalian evidence toward a potential role of LGRs as tumor suppressor genes (Scopelliti, 2014).

Altogether, these results demonstrate a novel paradigm in the regulation of intestinal homeostasis involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells, which may provide insights into other stem cell based systems (Scopelliti, 2014).

EFFECTS OF MUTATION

In the course of a genetic screen for flies that failed to expand their wings, five mutants were identified that formed a single complementation group that mapped to the vicinity of CG13419. Genomic sequencing of these mutants show that each has an independent single base pair change at CG13419. All have wing spreading and sclerotization defects when either homozygous or heterozygous over deficiencies for the region. Four mutants have sequence changes that are predicted to cause significant alterations of the mature protein structure. The burs^Z4410 mutation is a G to A conversion at the splice acceptor site for the second exon, which likely prevents normal splicing. This mutation would lead to read-through of the first intron, resulting in premature termination due to the presence of two consecutive stop codons. Since the mutation is located 194 bp from the translation initiation site, the resulting protein product would be comprised of the signal sequence and only 12 additional amino acids (one mutated) and is likely nonfunctional. Three other alleles have mutations within the coding region and either remove or introduce a cysteine residue and thus are likely to disrupt disulfide bridge formation, a key structural feature of cystine knot proteins. The burs^Z1091 allele results in a conversion of cysteine residue 82 to tyrosine, removing a cysteine that is conserved among all members of the mucin subfamily of cystine knot proteins. The burs^Z1140 mutation results in a conversion of threonine residue 97 to cysteine. The burs^Z5569 mutation results in a conversion of glycine residue 115 to cysteine. The fifth mutation, burs^Z2803, appears to be a mutation within the regulatory region. It results in a C to T change at -203 base pairs from the translation start site (Dewey, 2004).

The phenotypes of two bursicon mutants, burs^Z1091 and burs^Z5569, were examined in detail. Flies homozygous for either mutation, heteroallelic burs^Z5569/burs^Z1091 or heterozygous over deficiencies for the region, fail to spread their wings following eclosion and show a prolonged retention of the elongate abdomen shape characteristic of a newly eclosed fly. By the day after eclosion, their abdomens shorten but fail to taper into the normal adult shape, consistent with a failure to sclerotize properly. The two alleles differ in their effects on the timing of cuticle pigmentation and the expansion of the thoracic cuticle. The burs^Z5569 mutants are delayed in melanization, with only 40% completing it during the 3 hr following eclosion, but they inflate their thoracic cuticle normally. In contrast, burs^Z1091 mutants pigment normally but fail to complete thorax expansion, resulting in the postscutellar bristles remaining crossed. Flies that are heteroallelic for the two mutations (burs^Z5569/burs^Z1091) show characteristics of both mutants. They resemble burs^Z5569 mutants in being slow to pigment, with only 18% completing pigmentation within 3 hr of eclosion. However, they have crossed postscutellar bristles like burs^Z1091 homozygotes. Both bursicon mutations are recessive, displaying no wing expansion or sclerotization defects when in heterozygous combinations with complementing deficiencies, nonallelic mutants, or balancer chromosomes (Dewey, 2004).

The levels of bursicon produced by mutant larvae were determined using bursicon bioassays based on neck-ligated Sarcophaga bullata (Fraenkel, 1965; Kostron, 1995) or Drosophila (Baker, 2002). In the Sarcophaga bioassay, extracts of CNSs from control bw; st larvae stimulated tanning with an average score of 2.50 ± 0.18. By contrast, CNS extracts from burs^Z1091 mutant larvae yielded an average score of 0.27 ± 0.08, similar to that obtained with phosphate buffer controls. (In the Sarcophaga bioassay, bursicon activity below 10% of maximum activity cannot be detected. Thus, the level of bursicon in burs^Z1091 flies is less than 10% that of wild-type.) burs^Z5569 flies gave a low but measurable average score of 0.85 ± 0.63. Extracts from burs^Z5569/burs^Z1091 and burs^Z5569/Df (3R) e-GC3 yielded low scores (0.81 and 0.75, respectively), consistent with the hypothesis that the mutations in CG13419 are solely responsible for decreased bursicon bioactivity. Extracts from burs^Z5569/TM6B Tb (essentially burs^Z5569/+) larvae gave scores of 2.15 and 2.20; these are similar to those of the bw; st control (Dewey, 2004).

The blood of newly emerged flies was assayed for bursicon activity using a Drosophila test system (Baker, 2002). Injection of hemolymph from control bw; st flies into neck-ligated control bw; st or bursicon mutant flies resulted in tanning. In contrast, the hemolymph from homozygous burs^Z5569 or burs^Z1091 mutants of the same age failed to cause neck-ligated control flies to tan. These results are consistent with decreased bursicon activity in the mutants. It is not currently understood why burs^Z1091 fails to cause pigmentation in these bioassays despite the fact that when homozygous or in heterozygous combinations with deficiencies, burs^Z1091 mutants pigment normally. The amino acid changes in the two mutants may result in differences of their interaction with the receptor and/or in aspects of transport, metabolism, and stability of the mutant peptides (Dewey, 2004).

Genetic analysis of ecdysis behavior in Drosophila reveals partially overlapping functions of two unrelated neuropeptides

Ecdysis behavior allows insects to shed their old exoskeleton at the end of every molt. It is controlled by a suite of interacting hormones and neuropeptides, and has served as a useful behavior for understanding how bioactive peptides regulate CNS function. Previous findings suggest that crustacean cardioactive peptide (CCAP) activates the ecdysis motor program; the hormone bursicon is believed to then act downstream of CCAP to inflate, pigment, and harden the exoskeleton of the next stage. However, the exact roles of these signaling molecules in regulating ecdysis remain unclear. This study used a genetic approach to investigate the functions of CCAP and bursicon in Drosophila ecdysis. Null mutants in CCAP were shown to express no apparent defects in ecdysis and postecdysis producing normal adults. By contrast, a substantial fraction of flies genetically null for one of the two subunits of bursicon [encoded by the partner of bursicon gene (pburs)] show severe defects in ecdysis, with escaper adults exhibiting the expected failures in wing expansion and exoskeleton pigmentation and hardening. Furthermore, flies lacking both CCAP and bursicon show much more severe defects at ecdysis than do animals null for either neuropeptide alone. The results show that the functions thought to be subserved by CCAP are partially effected by bursicon, and that bursicon plays an important and heretofore undescribed role in ecdysis behavior itself. These findings have important implications for understanding the regulation of this vital insect behavior and the mechanisms by which hormones and neuropeptides control the physiology and behavior of animals (Lahr, 2012).

The fragmentary understanding of the regulation of ecdysis behavior indicates that it is controlled by a suite of neuropeptides and hormones that show complex hierarchical and reciprocal relationships, and in which a given neuropeptide (or hormone) may act on different targets or act in a combinatorial manner on a specific target with other neuropeptides (or hormones). This study isolated mutants null for CCAP and pburs to better define the functions of these genes and to investigate possible synergistic actions. It was surprising to find that animals lacking CCAP expressed normal pupal ecdysis behavior because CCAP is believed to be the key neuropeptide that controls ecdysis. For instance, application of CCAP to an isolated Manduca CNS will turn on the ecdysis motor program. Also, RNA interference (RNAi) of CCAP or its receptor, CCAPR-2, by injection of double-stranded RNA, causes arrest at ecdysis in Tribolium. Furthermore, because bursicon has previously only been associated with the regulation of postecdysis events following adult eclosion, it was were also surprising to discover that pburs-null mutants showed severe defects at pupation. Nevertheless, RNAi of both bursicon subunits as well as of its receptor (rickets, rk) cause a quantitative weakening of preecdysis behavior in Tribolium, and release of bursicon during Drosophila pupal preecdysis has recently been reported, suggesting a role in the control of early phases of the ecdysis sequence. Furthermore, it has been reported that interference of rk function in Drosophila causes defects at pupation, although the range of additional nonecdysial defects observed suggest that such manipulations interfered with other pathways, rendering the interpretation of these findings more difficult. In this regard, the results using animals mutant for pburs indicate that PBURS plays an important role, and that this role is primarily restricted to the correct execution of ecdysis behaviors (a role in postecdysis has not been investigated at this stage). A role for bursicon specifically at pupal ecdysis was recently uncovered by showing that defects at pupation, caused by the elimination of the retrograde signal needed for CCAP and PBURS expression in the CNS, could be partially rescued by specifically restoring pburs expression in the relevant neurons (Veverytsa, 2011; Lahr, 2012).

Although flies lacking CCAP were ostensibly entirely normal, it was possible to uncover a critical function for this peptide at ecdysis by examining pupation in animals lacking pburs function. Indeed, in this mutant background, eliminating CCAP caused an almost complete failure of ecdysis. This suggests that both CCAP and PBURS regulate ecdysis, with CCAP playing a minor role and PBURS playing a major role. The bases of CCAP and bursicon actions, however, remains unclear. The CCAP-expressing neurons in the ventral CNS consist of a pair of efferent neurons in segments T3-A4; homologous to cell 27s in other insects, and a pair of interneurons (CCAP_IN) in segments T1-A9; homologous to IN704 in other insects. In Manduca, addition of CCAP to an isolated CNS can activate and sustain the ecdysis motor program. The limited arborization of CCAP_E within the CNS would imply that this activational role would be subserved by CCAP_IN, and is consistent with the type of role that these neurons play after adult emergence; by contrast, CCAP_E neurons release bursicon into the hemolymph to first plasticize then harden and melanize the wings and exoskeleton, and play no behavioral role. Alternatively, the activation of ecdysis could be mediated by a pair of CCAP neurons in the subesophageal ganglion (SEG). At least at adult emergence, it is these neurons that command postecdysial behaviors, such as air-swallowing and wing inflation. However, both CCAP neurons in the SEG and the CCAP_INs express CCAP but not PBURS, and no ecdysial defects were detected in CCAP-null mutants. By contrast, recent findings show that reducing CCAP and bursicon expression from CCAP_Es causes severe defects in pupation, implying that these neurons may be key for the activation of ecdysis behavior. Since CCAP_Es have a sparse arborization within the CNS, these results would also imply that the activational roles of CCAP and bursicon could be indirect (Lahr, 2012).

While pupation requires activation of motor programs, changes in hemolymph pressure may also be essential for correct eversion of the head and the proper extension of legs and wings. Thus, it is also possible that CCAP's role in Drosophila ecdysis is at least in part based on its cardioactive function, instead of or in addition to a neural activation role. Indeed, CCAP is cardioactive in insects including Drosophila and, in addition to serving to better disperse coreleased neuropeptides and neurohormones, including bursicon, this cardioactive function may be necessary for the successful transformation into a pupa. The identification of the direct targets of CCAP and bursicon coupled with functional studies will be needed for the full understanding of the exact roles that these peptides play at pupation (Lahr, 2012).

Independent of the exact nature of their functions, the actions of CCAP and bursicon show the hallmark complexity of neuropeptide control of physiology and behavior. In addition to each of these molecules acting on different targets (e.g., bursicon, which activates ecdysis and also causes wing inflation and the hardening and pigmentation of the adult exoskeleton), it was shown that CCAP and bursicon act synergistically to control ecdysis behavior. Such convergence is seen in a number of peptide systems, and appears to be the basis for the integration of multiple signals and many time-independent signals. Such a situation occurs, for example, in the control of arousal, which depends on inputs related to food intake and satiation, as well as from inputs from the circadian clock. Another role for multiple peptidergic inputs may be to increase the precision and robustness of a response. For example, mammalian circadian clocks cause daily rhythms of locomotor activity to be expressed with a precision of ~1 min/d. The basis for this precision is not entirely understood, but is likely mediated by the action of multiple clock output neuropeptides, all of which can affect the pattern of activity/inactivity. Ecdysis likewise shows a very precise timing, and naturally occurring failures are extremely rare. Although this study has shown that CCAP is not essential for ecdysis in the laboratory, it may nevertheless provide a signal that, under particular conditions, is essential for the successful and seamless execution of the behavior. This signal may also vary in different insects, reflecting a bias toward one of several possible actions in organisms with different body plans. Thus, for instance, CCAP may primarily play a cardioactive role in some insects, whereas for others it may play a critical role in activating a motor program itself. It is hoped that future comparative work using insects with different developmental and anatomical constraints will help elucidate the logic behind such biases. In addition to such an approach, work in Drosophila and Tribolium has clearly shown that molecular genetics provides a unique tool to understand the essential as well as the redundant functions of every ecdysis neuropeptide and hormone. The combination of both approaches will shed light on the mechanism that enables insects to flawlessly complete a complex behavioral sequence almost regardless of conditions. It will also provide a useful model for understanding how neuropeptides control the physiology and behavior of all animals (Lahr, 2012).

EVOLUTIONARY HOMOLOGS

In an effort to characterize the insect molting hormone bursicon from the cockroach, Periplaneta americana, amino acid sequences with high identity of Cu,Zn-superoxide dismutase (SOD) of Drosophila virilis were identified. Antisera against a conserved region of SOD, and a sequence unique to Periplaneta SOD were produced and used to test whether bursicon might be a form of SOD. Western blots of one- and two-dimensional gels revealed that the dimeric form of SOD and bursicon have a similar molecular mass (30 kDa). The two proteins can be separated, however, according to their different isoelectric points. Bursicon is identified in two-dimensional gels by elution from four unique spots not labeled by the anti-SOD antisera. In sections of Periplaneta nerve cords, the antisera labeled glial material surrounding neuronal somata close to the neural sheath. Bursicon, however, is contained in unique cell pairs in the ganglia of the ventral nerve cord. These neurons were labeled with new antisera produced against novel sequences of one of the four above-mentioned bursicon active spots. The results show unequivocally that SOD and bursicon are distinctly different proteins. Furthermore, the anti-SOD antisera provided a tool to isolate and sequence bursicon (Kostron, 1999).

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two-dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon-containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon-containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two-dimensional gels. The 12-amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related (Honegger, 2002).

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum

Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, systemic RNA interference (RNAi) experiments were performed utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods (Arakane, 2008).

The earliest peptide signal for ecdysis behavior in Manduca so far identified is corazonin, which triggers the neuroendocrine cascade by inducing the release of ETH from the epitracheal glands. However, in Tribolium and also in other coleopteran species, corazonin is apparently absent because there has been no report of immunoreactivity with corazonin antiserum. Furthermore, no Tribolium sequences encoding this peptide or its receptor have been reported so far. Thus, the signal initiating ecdysis in this coleopteran must be something other than corazonin. In the case of the albino locust, which is believed to lack corazonin, there is no ecdysis deficiency reported, implying that corazonin may be lepidopteran-specific as a signal for ETH release (Arakane, 2008).

Severe deficiencies in preecdysis behavior were observed in Tribolium after treatment with either dseh, dseth, dsethr or dsethr-a. There were some occasional twitching-like D–V contractions in these insects, which may have been caused by incomplete suppression of the targeted mRNA. This deficiency in preecdysis behavior resulted in the failure of subsequent ecdysis behavior, which in turn resulted in failure to eclose and finally in death. The ETH signal has been found to be necessary and sufficient in Drosophila for both preecdysis and ecdysis behaviors. In Manduca, the sufficiency of ETH for inducing premature preecdysis and ecdysis behaviors also supports the conclusion that ETH is one of the earliest ecdysis-initiating molecules. This study also supports the notion of ETH being an early essential signal for ecdysis in Tribolium. In addition, dseth was found to cause deficiencies in larval and pupal ecdysis depending on the time of injection (Arakane, 2008).

Two subtypes of ETH receptors, A and B, arising from mutually exclusive alternative exon usage, are highly conserved in insects. Studies with Manduca and Drosophila showed that ethr-a is expressed mainly in numerous peptidergic cells in the CNS, while ethr-b is expressed in poorly-characterized interneuron cells. Exon-specific dsRNA in Tribolium showed that dsethr-a-treated insects had significantly fewer D–V contractions, a phenotype identical to that obtained following treatment with dseth or dseh. However, eclosion of insects injected with dsethr-b occurred normally, with no substantial reduction in preecdysial D–V contractions. Therefore, ethr-a, which activates downstream peptidergic signals, is a necessary component in Tribolium eclosion, whereas the role of ethr-b remains unclear. Switching from one behavior to the next within a certain time interval in the behavioral sequence had been thought to involve inhibitory neurons, it was not possible to determine whether premature ecdysis behavior occurred in the dsethr-b-injected Tribolium as a result of defects in inhibitory neurons (Arakane, 2008).

Positive feedback between EH and ETH has been found in Manduca. Release of ETH is triggered by corazonin as the initiator of the EH-ETH feedback loop. EH-associated positive feedback induces a massive release of ETH for the initiation of ecdysis motor patterns. However, a positive feedback loop was not found in Drosophila. Rather, Drosophila EH apparently acts downstream of ETH and is the factor triggering ecdysis behavior, a conclusion based on the timing of the cellular response of EH cells, which show Ca²⁺ elevation upon treatment with ETH during pupal ecdysis. Surprisingly, the EH-cell-knockout in Drosophila resulted in only a partial impairment during adult eclosion, with a significant proportion of the insects dying before pupation. In Tribolium, EH was required for early preecdysis behavior. Thus, the ETH-EH feedback loop, if it occurs, probably occurs during preecdysis in Tribolium, as it does in Manduca (Arakane, 2008).

In Drosophila, the ccap null mutant did not show any abnormality during development or ecdysis, whereas ccap-cell ablation resulted in deficiencies in both pupal and adult ecdysis. Therefore, it was concluded that other neuropeptides, which are co-expressed in the CCAP cells, are probably responsible for the phenotypes of the ccap cell-knockout. Thus, the role of CCAP in Drosophila ecdysis remains unclear. The neuropeptides co-expressed in the CCAP cells with presumed functions in ecdysis are bursicon, partner of bursicon and myoinhibitory peptide (Arakane, 2008).

In contrast to Drosophila, ccap RNAi in Tribolium resulted in a lethal arrest during ecdysis. The ecdysis deficiency was associated with significantly weaker behaviors, including reverse-bending, wing air-filling and A–P contraction, whereas these insects underwent normal preecdysis behavior. The dsccapr-2 treatment resulted in the same phenotype as that of dsccap, whereas dsccapr-1 treatment did not produce any abnormalities. Therefore, CCAP and CCAPR-2 are in the signaling pathway for ecdysis behavior, while the function of CCAPR-1 remains unknown (Arakane, 2008).

The bur and pbur genes in Tribolium form a tandem pair in the genome, separated by only ~1.3 kb. This arrangement is similar to the bur/pbur gene structure in the honeybee. Previously, it was proposed that the honeybee bur/pbur gene consisted of one long open reading frame encoding a multi-domain protein including both bur and pbur. Subsequently, however, different transcription units for bur and pbur were reported. Using RT-PCR this study determined that Tribolium bur and pbur are probably separate transcription units. Results from gene-specific RNAi for both bur and pbur support the hypothesis that two different transcription units exist. In addition, whereas mosquito bur was found to undergo trans-splicing, this study found that the Tribolium bur and pbur genes contain complete open reading frames, excluding the possibility of trans-splicing (Arakane, 2008).

A heterodimeric complex Bur/pBur consisting of the products of the bur and pbur genes is a cysteine knot family hormone that has been reported to initiate two different functional activities in Drosophila, namely cuticle tanning and wing expansion after adult eclosion. Drosophila mutants for bur and receptor mutant rickets (rk) showed deficiencies in both tanning and wing expansion (Arakane, 2008).

This study discovered that treatments with dsbur, dspbur or dsrk all produced similar postecdysis defects, namely weak postecdysis activity, wrinkled elytra and a failure to retract the hindwing, but none of these caused lethality within the observation time of 2–3 weeks after eclosion. In Drosophila, bursicon induces wing cell death and wing expansion after eclosion. The wrinkled elytra and the deficiency in proper folding of the hindwing after RNAi in Tribolium may be equivalent to the Drosophila phenotype. Interestingly, RNAi of these genes resulted in significantly diminished strengths in preecdysis behavior. The data imply that bur/pbur and their putative receptor rk in Tribolium are involved in the regulation of preecdysis behavior, and even more in postecdysis behavior. An additional unique phenotype was found only in insects injected with dsbur, which exhibited weaker A–P contractions during ecdysis and consequently an extended duration for completion of the shedding of the exuvium. These observations suggest an unknown but separate function for bursicon in addition to its role as a component of dimeric Bur/pBur acting through its receptor Rickets. Alternatively, the phenotypic variation could have been caused by different dosages of remaining transcripts in RNAi or by stability of the protein that had been produced earlier (Arakane, 2008).

Perhaps the most interesting observation in this study is that normal tanning occurs in beetles subjected to RNAi for the group of genes encoding the neuropeptides described in this study. Maturation of the cuticle is a gradual process of pigmentation and sclerotization during the first five days after eclosion. A recent study involving Drosophila has shown that bursicon acts through phosphorylation of tyrosine hydroxylase, which catalyzes an early step of catecholamine production for cuticle tanning. It has been shown previously that RNAi of Tribolium laccase 2, which is a phenoloxidase downstream of tyrosine hydroxylase in the same metabolic pathway, suppressed cuticle tanning. This result indicated that a similar cuticle tanning pathway exists in both Drosophila and Tribolium. It is concluded, therefore, that the Bur/pBur signaling pathway is required for proper wing expansion and folding in Tribolium but not for tyrosine hydroxylase/laccase-mediated tanning. Recent study in the silkworm also reported that RNAi of bur found no distinct tanning phenotype, while a deficiency in wing expansion was observed. In addition, the regulation of cuticle tanning in Tribolium appears to be different from that of Drosophila, even though the tanning pathway itself is probably conserved (Arakane, 2008).

This has been a study of key peptidergic signaling systems for insect ecdysis in T. castaneum, representing a more basal holometabolous order (Coleoptera) relative to the species of Lepidoptera and Diptera studied previously. RNAi in Tribolium followed by behavioral analysis revealed differences in the roles of EH, CCAP and bursicon compared to those found in Drosophila. Both ETH and EH are necessary for preecdysis and ecdysis behaviors in Tribolium, while an essential role of EH has not been found in Drosophila. CCAP is necessary for ecdysis behavior in Tribolium, whereas the Drosophila ccap null mutant shows normal ecdysis. In Tribolium Bur/pBur is necessary for postecdysis behavior, including wing expansion and folding, whereas, unlike the case in dipterans, it does not have a role in cuticle tanning (Arakane, 2008). Bur/pBur signaling is involved in preecdysis behavior. Only bursicon appears to have an additional role in ecdysis behavior in Tribolium (Arakane, 2008).

The differences in the precise roles of each peptidergic component among Tribolium, Drosophila and Manduca in controlling innate ecdysis behavior and cuticle tanning can be interpreted as a consequence of evolution. The loss of essential roles for EH and CCAP as well as a gain in function for bursicon in Drosophila may be associated with modifications of the requirements of those neuropeptidergic signals in these processes, whereas the more ancestral Tribolium and possibly Manduca strictly require EH and CCAP signaling systems. A comparative analysis of the functions of peptidergic signals from additional taxa will provide further insights into the evolution and regulation of ecdysis and tanning in the Ecdysozoa (Arakane, 2008).

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date revised: 4 April 2022

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