InteractiveFly: GeneBrief

Crustacean cardioactive peptide: Biological Overview | Regulation | Developmental Biology | Effects of mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. Drosophila bearing targeted ablations of CCAP neurons (Ccap-KO animals) have been used to investigate the role of Ccap in the execution and circadian regulation of ecdysis. Ccap-KO animals show specific defects at ecdysis, yet the severity and nature of the defects vary at different developmental stages. The majority of Ccap-KO animals die at the pupal stage from the failure of pupal ecdysis (pupation), whereas larval ecdysis and adult eclosion behaviors show only subtle defects. Interestingly, the most severe failure seen at eclosion appears to be in a function required for abdominal inflation, which could be cardioactive in nature. Although Ccap-KO populations exhibit circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) is abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of Ccap as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila (Park, 2003).

Ecdysis is a complex yet stereotyped behavior whose timing must be precisely coordinated with the molting cycle such that it is turned on only when the old cuticle is sufficiently resorbed that it can successfully be shed. In addition, the timing of some ecdyses, typically that to the adult (adult ecdysis or eclosion), can be under the control of the circadian clock. While molting (the production of the new cuticle) is regulated by the ecdysteroid class of steroid hormones, the timing as well as the execution of ecdysis behavior is controlled by a number of neuropeptides: Eclosion hormone (EH), Ecdysis triggering hormone (ETH, and associated Pre-ecdysis triggering hormone, PETH: see Drosophila ETH), and Crustacean cardioactive peptide (Ccap) (reviewed by Ewer, 2002). Of these, Ccap is believed to be the neuropeptide that turns on the ecdysis motor program. In addition to a role in the execution of ecdysis, strong circumstantial evidence suggests that Ccap may be one of the factors that regulate the circadian timing of adult ecdysis (eclosion). For example, the Lark RNA-binding protein has been implicated in the circadian control of Drosophila eclosion (Newby, 1993), and it is localized (McNeil, 1998; Zhang, 2000) preferentially in the cytoplasm of CCAP neurons (Park, 2003 and references therein).

Although this model for the hormonal control of ecdysis is consistent with most of the available data, a number of observations suggest that the control of this behavior occurs via a more complicated mechanism. For instance, adult ecdysis still occurs in Drosophila lacking EH neurons (McNabb, 1997). Likewise, although the genetic deletion of the gene encoding ETH causes most animals to die at the first larval ecdysis (Park, 2002a), many of these animals still display ecdysis-like behavior at the end of this molt (Park, 2003).

The complex phenotypes of these variants also raises the possibility that the role of Ccap in the control of ecdysis may not be as simple as currently proposed. This study has used Drosophila to investigate the roles of Ccap in the control and circadian regulation of ecdysis. Genetic ablation of the Ccap neurons causes defects at ecdysis. However, the type of defects observed at the ecdyses to different developmental stages as well as the severity of these defects suggest that the role of Ccap in the control of ecdysis varies during postembryonic development. In addition, although populations of flies lacking Ccap neurons exhibit a circadian rhythmicity of eclosion, the daily timing of eclosion events is abnormal in these animals, implying a modulatory role for Ccap in the circadian control of this behavior (Park, 2003).

Strong circumstantial evidence implicates CCAP in the control of ecdysis behavior. In the moth Manduca, in vitro experiments using isolated abdominal central nervous systems suggest that CCAP is required for turning off the pre-ecdysis motor program (Gammie, 1997b) and turning on that for ecdysis (Gammie, 1997b; Zitnan, 2000). The phenotypes caused by ablating Ccap neurons in Drosophila are, overall, consistent with this model. Indeed, both larval and pupal pre-ecdysis are longer than normal in KO animals, in agreement with an inhibitory action of Ccap on the expression of this preparatory behavior. In addition, the lack of Ccap neurons causes the complete failure of pupal ecdysis (pupation), strongly suggesting that the Ccap neuropeptide is essential for turning on ecdysis behavior (Park, 2003).

Although Ccap neurons are essential for pupation, the ecdysis motor program of both larval and adult KO animals appears qualitatively normal, implicating additional mechanisms in the control of these behaviors. Ccap may play a minor role at these times or, alternatively, other neuropeptides may compensate for the loss of Ccap. Irrespective of the exact mechanism, the results of this study strongly suggest that other pathways, independent or compensatory, exist, which control the expression of these motor programs. To date the only gene that is known to be essential for ecdysis is the ETH gene, and flies carrying the null ETH alleles die at the first larval ecdysis. However, the ETH peptides are believed to act upstream of Ccap (reviewed by Ewer, 2002), and in Drosophila, ETH is released before Ccap at larval and pupal ecdysis, consistent with this hypothesis. Thus, it is unlikely that the ETH peptides act in parallel with Ccap or can compensate for it absence. In addition, the findings that the lack of Ccap does not cause larval lethality argue against a simple linear pathway in which the essential function of ETH is to cause release of Ccap leading to the initiation of the ecdysis motor program. EH is also believed to act upstream of Ccap (reviewed by Ewer, 2002). However, the exact role of EH in the control of ecdysis is currently unclear, since EH KO animals are usually able to ecdyse, although their behavior is somewhat disorganized. An examination of the ecdysis of animals lacking both EH and Ccap neurons compared with that of Ccap-KO and EH KO animals will reveal the extent to which EH can compensate for the lack of Ccap, and vice versa (Park, 2003).

In addition to compensatory mechanisms, other mechanisms may contribute to the varying importance of Ccap at different ecdyses. For instance, subsets of Ccap neurons may participate at some ecdyses but not at others. In the abdominal CNS of the Manduca for example, 2 pairs of CCAP-immunoreactive neurons up-regulate the second messenger cGMP at larval ecdysis, whereas only one pair does so at pupal and adult ecdysis (Ewer, 1997). Since this cGMP response likely increases the excitability of the Ccap neurons [it is known to do so for the thoracic set (Gammie, 1997a)], this change in the pattern of cGMP expression could change the relative participation of the different Ccap neurons at each ecdysis. It is not known if this sort of mechanism applies to Drosophila, since no cGMP response is detected in Ccap neurons at any ecdysis in this species (Ewer, 1996; Baker, 1999). Nevertheless, the differential activation of a subset of peptidergic neurons at different times in development could provide a mechanism for modifying the extent of the participation of these neurons in different behavioral or developmental contexts. Alternatively, the role of Ccap may change during postembryonic development because of changes in the expression of Ccap receptors. Although the Ccap receptor has not been conclusively identified (but see Park, 2002b), the completion of the Drosophila genome sequence and its subsequent analyses (Brody, 2000; Hewes, 2001) have produced a list of potential candidates (Park, 2003).

The most dramatic feature of KO animals at adult eclosion is not in the expression of the ecdysis motor program itself, but a function that may be cardioactive in nature. It may be that the Ccap neurons are important for increasing hemolymph pressure, and CCAP is known to be a cardioactive peptide in insects (see Dircksen, 1998) including Drosophila (Nichols, 1999), and to be released at eclosion in Manduca. Alternatively, the defect may be in fluid homeostasis. In crabs, for instance, the shedding of the old carapace is preceded by a massive release of hyperglycemic hormone (HH) which causes a swelling of the body via an anti-diuretic mechanism (Chung, 1999). CCAP is also released at this time (Phlippen, 2000) and could regulate HH release. Regardless of the bases for the defects observed in eclosing KO animals, their phenotype suggests that maintaining a high internal body pressure is critical for adult eclosion, and implicates the Ccap neurons in this process (Park, 2003).

Features of lark gene expression in the Ccap neurons, as well as the potential for synaptic contact between Ccap and clock neurons suggests that Ccap may play a role in mediating the circadian control of adult eclosion. Although the rhythmic eclosion profile of Ccap-KO populations shows that Ccap neurons are not essential for the circadian gating of eclosion, the distribution of eclosion events in this population indicates that these neurons modulate the gating process. This modulation may occur via a direct connection with clock neurons or other peptidergic neurons (e.g., those expressing PDF), and the anatomy of Ccap neurons in the brain is consistent with this hypothesis. The robust circadian rhythmicity of Ccap-KO populations indicates that there are multiple (and potentially redundant) cellular pathways mediating the output of the clock (Park, 2003).

Several lines of evidence suggest that Ccap neurons mediate the effects of light on eclosion, indirectly via the EH neurons. In Manduca, strong circumstantial evidence suggests that CCAP acts downstream of EH (reviewed by Ewer, 2002). In Drosophila, Ccap release occurs after EH release at larval ecdysis (A. C. Clark, M. del Campo, and J. Ewer, unpublished data cited in Park, 2003), suggesting that the same relationship may exist in the fly. Importantly, EH KO and Ccap-KO animals both show an altered response to the light-on signal, and recent evidence suggests that light can cause a premature release of EH (S. McNabb and J. W. Truman, personal communication to Park, 2003). Thus, it is possible that certain Ccap neurons mediate the light-on response that is channeled through the EH neurons (Park, 2003).

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).

CCAP regulates feeding behavior via the NPF pathway in Drosophila adults

The intake of macronutrients is crucial for the fitness of any animal and is mainly regulated by peripheral signals to the brain. How the brain receives and translates these peripheral signals or how these interactions lead to changes in feeding behavior is not well-understood. This study discovered that 2 crustacean cardioactive peptide (CCAP)-expressing neurons in Drosophila adults regulate feeding behavior and metabolism. Notably, loss of CCAP, or knocking down the CCAP receptor (CCAP-R) in 2 dorsal median neurons, inhibits the release of neuropeptide F (NPF), which regulates feeding behavior. Furthermore, under starvation conditions, flies normally have an increased sensitivity to sugar; however, loss of CCAP, or CCAP-R in 2 dorsal median NPF neurons, inhibited sugar sensitivity in satiated and starved flies. Separate from its regulation of NPF signaling, the CCAP peptide also regulates triglyceride levels. Additionally, genetic and optogenetic studies demonstrate that CCAP signaling is necessary and sufficient to stimulate a reflexive feeding behavior, the proboscis extension reflex (PER), elicited when external food cues are interpreted as palatable. Dopaminergic signaling was also sufficient to induce a PER. On the other hand, although necessary, NPF neurons were not able to induce a PER. These data illustrate that the CCAP peptide is a central regulator of feeding behavior and metabolism in adult flies, and that NPF neurons have an important regulatory role within this system (Williams, 2020).

This study demonstrates that 2 residual CCAP neurons not only signal for food intake but also have a role in regulating metabolism, where they are important for maintaining triglyceride levels. CCAP signaling activates an NPF pathway for proper sensing of sugars for food intake. Of note, flies lacking CCAP, or CCAP-R in 2 dorsal median P1 NPF neurons, are not able to distinguish nutritive from nonnutritive sugar. However, CCAP signaling to these P1 NPF neurons is not sufficient for the NPF feeding phenotypes, as knocking down CCAP-R specifically in these neurons or inhibiting these 2 neurons using the inward-rectifying channel Kir2.1 was not able to recapitulate the phenotypes. This hints at other CCAP- and NPF-regulated neurons being involved in the control of feeding behavior. One possibility is the peripheral L1-I NPF neurons, as they did react to starvation by increasing NPF protein levels and exhibited increased NPF protein levels when CCAP neurons were activated. When flies sense palatable food, a reflexive behavior known as the proboscis extension reflex is initiated. This study shows that CCAP is both necessary and sufficient to induce this reflex. Moreover, dopaminergic neurons are also sufficient to induce this response, but not NPF neurons. Thus, this study has identified CCAP as a possible key node in regulating feeding behavior (Williams, 2020).

Previously it was shown that under acute starvation one of the Drosophila NPY homologs, NPF, initiates a response that activates dopaminergic signaling, leading to the sensitization of gustatory neurons (Gr5a) toward sugar taste. This study shows that Drosophila CCAP regulates the activity of the 2 dorsal median P1 NPF neurons and that this is sufficient to control food intake. First, CCAP neurons project toward the 2 dorsal median P1 NPF neurons. Second, knocking down CCAP expression, or CCAP-R expression in the dorsal median P1 NPF neurons, increases NPF expression under ad libitum conditions (CCAP-R knockdown), as well as in response to starvation (both CCAP and CCAP-R). It is interesting that loss of CCAP-R already significantly influences NPF expression under conditions where flies are fed ad libitum. Possibly, NPF is being released at low levels even under fed conditions. It is known that NPF also regulates sleep and the reward system. CCAP may signal upstream of NPF to regulate sleep and reward as well. This possibility should be tested in the future. Furthermore, activating CCAP neurons using thermogenetics was sufficient to reduce NPF expression in the dorsal median P1 NPF neurons, indicating the neurons were more active. On the other hand, activating CCAP neurons increased NPF expression in the dorsal lateral L1-I neurons, indicating that activation of CCAP neurons inhibited these NPF-expressing neurons. This increase of NPF in the peripheral L1-I neurons was also observed when flies were starved. From these data, it is concluded that activating CCAP neurons in turn activates 2 dorsal median NPF neurons, leading to sugar sensitization. Tge inability to recapitulate the CCAPexc7 feeding phenotypes by knocking down CCAP-R expression specifically in the dorsal median P1 NPF neurons, or inhibiting these 2 neurons using the inward-rectifying channel Kir2.1, may indicate that the peripheral L1-I NPF neurons also play an important role in regulating food intake. More work is needed to understand CCAP's possible regulation of the peripheral neurons, as loss of CCAP had no influence on NPF protein levels in these neurons (Williams, 2020).

This study found that CCAP neurons were not only necessary, but also sufficient, to induce the PER. Previously, by the use of optogenetics, it was determined that Gr5a neurons were sufficient to induce the PER. On the other hand, although NPF neurons and dopaminergic neurons were determined to be necessary for a proper PER when flies were presented with varying concentrations of sugar, it was not established whether they were sufficient. Using optogenetics, this study determined that while dopaminergic neurons (ple-GAL4) were able to induce a PER, NPF neurons (NPF-GAL4 or R64F05-GAL4) were not sufficient (Williams, 2020).

Interestingly, adult flies lacking the CCAP peptide had significantly lower triglyceride levels. This was not observed when CCAP-R was specifically knocked down in NPF neurons. In order to maintain homeostasis, the brain must process extrinsic and intrinsic information. In Drosophila, different peptides have been shown to regulate these signals, such as diuretic hormone 44 (Dh44), corazonin (Crz), allatostatin A (AstA), and SIFamide (SIFa). Furthermore, similar to mammals, insulin-like peptides and a glucagon-like hormone (AKH) are also involved in regulating feeding behavior. Interestingly, Dh44 was shown to be necessary for the fly to sense postprandial nutritional information, and this study showed that flies lacking either CCAP or CCAP-R in NPF neurons were unable to determine between nutritional and nonnutritional sugars. That said, it must be mentioned this experiment only lasted 1 h and longer times may be necessary to truly understand if CCAP is involved in regulating postprandial nutritional signals. Another possibility is that CCAP neurons regulate Crz signaling. Crz is a Drosophila peptide related to mammalian gonadotropin-releasing hormone. Activation of Crz-expressing neurons was reported to reduce triglyceride levels, while loss of Crz regulation of insulin-producing cells leads to increased triglyceride storage, suggesting that Crz signals to decrease energy reserves. It is possible that CCAP signals to inhibit Crz in order to control the decrease in energy reserves under starvation conditions. Furthermore, loss or activation of SIFa neurons in adult flies produced feeding phenotypes very similar to when CCAP signaling is inhibited or activated, meaning there could be an interaction between SIFa and CCAP as well (Williams, 2020).

In summary, these experiments identify 2 CCAP peptidergic neurons as being required to induce feeding behavior via the NPF pathway in adult Drosophila. It is suggested that CCAP-expressing neurons regulate feeding behavior and are necessary for the proper sensing of sugars, while also regulating triglyceride levels. Continued studies of these 2 CCAP neurons, their neuronal network, as well as how they regulate feeding behavior and metabolism may help in understanding of satiety control and how peripheral physiological signals are translated into behavioral changes by the brain (Williams, 2020).

REGULATION

Promoter Structure

In order to establish that a Ccap-GAL4 transgene accurately reproduces the expression of the Ccap gene, it was first used to drive expression of the reporter lacZ, and the spatial expression of the reporter was compared to that of Ccap. In Drosophila, the Ccap peptide is consistently expressed in 2 pairs of neurons in the brain, 5 pairs in the subesophageal ganglion, 1-2 pairs in at least 8 ganglia of the ventral nervous system (ventral nervous system), as well as in 2 pairs of strongly immunoreactive descending axons, one lateral and one medial (see Ewer and Truman, 1996). No evidence of changes in the number of neurons that express Ccap-IR during postembryonic development was found except following adult eclosion, when there is a precipitous decrease in the number of Ccap neurons due to their elimination by programmed cell death (Draizen, 1999). Thus, unlike the situation in Manduca (Davis, 1993; Loi, 2001), no CCAP immunoreactive neurons appear to be added to the pattern that is established by the 1st instar larval stage (Park, 2003).

GAL4 fusions bearing the -516 to +39 bp fragment of 5' Ccap DNA faithfully reproduce the temporal and spatial pattern of Ccap expression. Thus, in all cases examined, neurons that are Ccap immunoreactive are also ß-gal immunoreactive, and vice versa. The stages examined included 1st instar (0- to 2-hour, 6- to 8-hour and 21- to 24-hour-old 1st instars), mid- and late-2nd instar, pharate and wandering 3rd instar larvae, pharate pupae, pharate adults, and 6-day-old adults (late 2nd instar). All three independent transformant lines bearing this construct showed indistinguishable patterns of expression (Park, 2003).

Physiological effects of Ccap

The effect of crustacean cardioactive peptide on Drosophila heart rate was measured in the animal and in a tissue preparation. Crustacean cardioactive peptide increases in vivo basal heart rate 1%, 6%, and 19% and increases in vitro basal heart rate 52%, 25%, and 35% in larvae, pupae, and adults, respectively. In the tissue preparation, the acceleratory period is followed by decreased in vitro heart rates of 42%, 16%, and 13% in larvae, pupae, and adults, respectively. The effects observed in the animal and tissue and in larvae, pupae, and adults suggest that Drosophila crustacean cardioactive peptide cardiac signaling is modulated and developmentally regulated (Nichols, 1999).

A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles

At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. To elucidate neural substrates underlying the ecdysis sequence, neurons expressing ETH receptors (ETHRs) have been identified in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. These findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles (Kim, 2006a).

Analysis of the pupal ecdysis behavioral sequence: In Drosophila, pupal ecdysis is preceded by pupariation, whereby the prepupa contracts its body into a barrel shape to form the puparium composed of the old larval cuticle. The underlying new pupal cuticle then separates from the puparium during pupal ecdysis ~12 hr later. The stereotypic nature of pupal ecdysis and reliable developmental markers make it a favorable model for the behavioral analysis and neural imaging (Kim, 2006a).

Pupal ecdysis consists of three centrally patterned behavioral subunits performed sequentially: pre-ecdysis (~10 min), ecdysis (~5 min), and postecdysis (~60-70 min). The behavioral sequence was examined through the semitransparent puparium ('puparium-intact'), but it was found that the puparium obscures and places constraints on some movements. This made it particularly difficult to discriminate differences in abdominal swinging movements during ecdysis and postecdysis. Therefore, a 'puparium-free' preparation was used by surgically removing the puparium immediately after pre-ecdysis onset. The improved visibility and room for movement in this preparation allowed for a more complete analysis of natural and ETH-induced behavior. The following description of the natural pupal ecdysis sequence resulted from comparison of behaviors observed in both puparium-intact and puparium-free prepupae (Kim, 2006a).

Pre-ecdysis: About 5 min after in vivo ETH release, preecdysis commences with the abrupt appearance of an air bubble at the posterior end of the prepupa (time zero). Pre-ecdysis involves anteriorly directed rolling contractions along the lateral edges of the abdomen, alternating on the left and right sides of the animal. These contractions move the air bubble anteriorly to separate pupal cuticle from the puparium. This behavior is completed within ~10 min and is followed by ecdysis behavior. Preecdysis behavior is the same in puparium-intact and puparium-free animals (Kim, 2006a).

Ecdysis: In higher Diptera including Drosophila, the incipient adult head develops within the prepupal thorax. During pupal ecdysis, head eversion results from lateral swinging movements of the abdomen occurring along with anteriorly directed peristaltic contractions. In puparium-intact preparations, head eversion occurs ~1 min after the onset of ecdysis swinging and is completed within ~5s. After completion of head eversion, ecdysis contractions continue for ~15 min, facilitating expansion of wing pads and legs to their final size. The frequency of ecdysis swinging (~5 swings/min) decreases markedly after head eversion (~2 swings/min). In puparium-free animals, head eversion occurs sooner, and the duration of ecdysis behavior lasts only ~5 min. Later in ecdysis, anteriorly directed swinging contractions are often interrupted by posteriorly directed ones, indicating a transition to postecdysis (Kim, 2006a).

Postecdysis: Postecdysis behavior consists of two behavioral subroutines: postecdysis swinging and stretch-compression movements of the abdomen. Postecdysis swinging occurs along with posteriorly directed peristaltic contractions and alternates with longitudinal movements of the abdomen, referred to as 'stretch-compression.' The frequency and intensity of postecdysis contractions wane gradually until they are detected mainly in the anterior part of the abdomen; they cease w100 min after pre-ecdysis onset. Postecdysis behavior concludes with compression of the pupa at the posterior end of the puparium (Kim, 2006a).

ETH release coincides with initiation of the ecdysis sequence: To confirm the role of ETH in initiation of the pupal ecdysis sequence, its release from endocrine Inka cells was monitored in vivo by using time-lapse EGFP fluorescence imaging in pharate pupae (prepupae) carrying the chimeric transgene 2eth3-egfp. In this transgenic fly, EGFP is expressed as part of a fusion protein with the ETH propeptide precursor, and loss of EGFP fluorescence indicates ETH release. Because pharate pupae generally are immobile and Inka cells are located immediately below the semitransparent puparium, in situ imaging of Inka cell in intact pharate pupae is feasible. Two to three Inka cells were monitored simultaneously in each experiment (Kim, 2006a).

Depletion of ETH-EGFP occurs in about 50% of monitored Inka cells shortly before pre-ecdysis onset (time zero). The time course of secretory activity for each Inka cell was variable. The mean value for the timing of ETH release was 4.5 min prior to pre-ecdysis onset, and the duration of ETH secretion was 4.4. In contrast, 40% of Inka cells showed no sign of secretory activity by pre-ecdysis onset. After initiation of pre-ecdysis contractions, it was usually impossible to continue monitoring loss of EGFP fluorescence as a result of movement artifacts. All Inka cells are depleted of ETH-EGFP by the end of ecdysis sequence (Kim, 2006a).

Injection of ETH induces the ecdysis sequence: Because ETH release coincides with onset of pupal preecdysis, it was of interest to determine whether ETH injection would trigger the pupal ecdysis sequence. The Drosophila gene eth encodes a precursor producing one copy each of two peptides, ETH1 and ETH2, which share similar structure and biological activity. In vivo experiments were carried out primarily in puparium-free preparations (Kim, 2006a).

Injection of ETH1 alone into pharate pupae (~1-2 hr prior to natural ecdysis) induced within 1-3 min strong pre-ecdysis contractions followed by ecdysis and postecdysis contractions sequentially. ETH-induced pre-ecdysis showed a strong dose dependence, with higher doses inducing shorter pre-ecdysis duration and higher frequency of contractions. Similar but somewhat less pronounced dose-dependent effects were observed during ecdysis behavior, whereas the frequency of postecdysis contractions showed little or no dose dependence during the first 10 min of behavior (Kim, 2006a).

Injection of ETH2 was less efficacious for induction of the behavioral sequence compared to ETH1. ETH2 generated prolonged pre-ecdysis behavior lasting to 50 min or more, but no ecdysis behavior. In contrast, injection of the same dose of ETH1 (0.4 pmol) produced the complete behavioral sequence consisting of pre-ecdysis, ecdysis, and postecdysis. Higher doses of ETH2 (20 pmol) generated a behavioral sequence comparable to that induced by 4 pmol ETH1 in terms of pre-ecdysis duration and frequency of contractions. Behaviors after injecting a cocktail of ETH1 and ETH2 (0.4 pmol of each peptide) were also examined. Because the two peptides are processed from the same precursor, it is likely that these peptides are coreleased under natural conditions. The duration of the behavioral sequence induced by injection of the cocktail was similar to the naturally occurring sequence or one induced by 0.4 pmol ETH1 alone. It is estimated that a 0.4 pmol ETH injection into a prepupa w10 hr after puparium formation results in a concentration of ~300 nM in vivo (Kim, 2006a).

ETH receptors are expressed in diverse ensembles of peptidergic neurons: ETH acts directly on the CNS to initiate the ecdysis behavioral sequence in moths and flies via unknown signaling pathways within the CNS. A starting point for elucidation of these downstream signaling pathways is identification of primary neuronal targets of ETH. The ETH receptor gene (CG5911), first identified in Drosophila, encodes two G protein-coupled receptors, ETHR-A and ETHR-B, via alternative splicing. In situ hybridization was used for identification of central neurons expressing ETHR-A and ETHR-B by using DNA probes specific for each receptor subtype. ETHR-A and ETHR-B transcripts were located in mutually exclusive populations of neurons distributed throughout the CNS, suggesting that two subtypes of ETH receptors likely mediate different functions. Further analysis revealed that most ETHR-A neurons are peptidergic. Neurons expressing ETHR-B have not been identified thus far (Kim, 2006a).

Multiple ensembles of ETHR-A neurons are classified according to specific neuropeptides they express. Peptides expressed in these ensembles were identified by using GAL4 transgenes under control of neuropeptide promoters to drive UAS-GFP or UAS-GCaMP expression (GAL4::GFP or GAL4::GCaMP). Expression of neuropeptides in these cells was confirmed by combining immunohistochemical staining and in situ hybridization. The first ETHR-A ensemble comprises six pairs of lateral abdominal neurons producing kinin, also known as drosokinin. These cells project axons posteriorly along the lateral edge of the neuropile and then turn anteriorly along the midline of ventral nerve cord, where they arborize and form possible central release sites. Axons of these cells also exit the CNS through nerve roots, suggesting peripheral kinin release. The second ETHR-A ensemble contains three pairs of ventrolateral FMRFamide neurosecretory cells (Tv1-3 or T1-3) in the thoracic neuromeres TN1-3. These cells project axons into the dorsomedial neurohemal organs (NHOs) specialized for peptide release into the hemolymph (Kim, 2006a).

The third class of ETHR-A neurons comprises the eclosion hormone (EH)-producing VM neurons in the brain, which project one axonal branch anteriorly into the neurohemal ring gland and a second posteriorly along the dorsal midline of the entire ventral nerve cord. The fourth ETHR-A ensemble is composed of paired dorsolateral neurons producing CCAP, MIPs, and bursicon in subesophageal, thoracic, and abdominal neuromeres (SN1-3, TN1-3, AN1-7, respectively). These cells are likely homologs of moth neurons 27/704, on the basis of their anatomy, peptide coexpression profile, and functional roles during pupal ecdysis. These neurons are referred to as Drosophila neurons 27/704 and them were subdivided on the basis of peptide coexpression. In AN1-4, CCAP is colocalized with MIPs and the heterodimeric peptide hormone bursicon (composed of burs and pburs subunits). In TN2-3 and AN5- 9, CCAP is colocalized with burs, but pburs is not expressed in these neurons. Finally, CCAP is colocalized with MIPs in large paired neurons of AN8,9, but ETHR-A expression has not been confirmed in these cells. The presence of MIP mRNA in abdominal neurons 27/704 was further confirmed by in situ hybridization (Kim, 2006a).

Ca2+ imaging of primary ETH targets in transgenic flies: Having shown that ETH receptors occur in diverse ensembles of peptidergic neurons, it was asked whether these cells are activated by ETH and whether this activity coincides with specific behavioral steps of the ecdysis sequence. Calcium dynamics in each group of ETHR-A neurons was monitored by driving expression of the GFP-based Ca2+ sensor, GCaMP [23, 24], in genetically defined sets of neurons with the binary GAL4/UAS system. Ca2+ elevation induces a conformational change of GCaMP, increasing its GFP fluorescence. Using optical imaging of GFP fluorescence, [Ca2+]i dynamics of ETHR neurons were monitored, and these events were associated with each behavioral phase induced by ETH (Kim, 2006a).

An abundance of evidence indicates that the ecdysis behavioral sequence in insects is centrally patterned. In particular, the onset and duration of each behavior in the sequence (pre-ecdysis I, pre-ecdysis II, ecdysis) is the same whether observed in vivo or as fictive behavior recorded from the isolated CNS in vitro. On the basis of this evidence, [Ca2+]i dynamics of ETHR-A neuron ensembles of the isolated CNS were associated with behaviors observed in puparium-free preparations (Kim, 2006a).

FMRFamide neurons and their neurohemal endings become active early in pre-ecdysis: [Ca2+]i levels were monitored in ETHR-A/FMRFamide Tv neurons by preparing transgenic flies doubly homozygous for FMRFa-GAL4 and UAS-GCaMP. Prior to ETH1 exposure (4-6 hr prior to ecdysis), Tv cell bodies and neurohemal endings in the dorsomedial NHO exhibit low levels of basal GCaMP fluorescence (Kim, 2006a).

Exposure of the CNS to ETH1 (600 nM) elicits robust increases in calcium-associated fluorescence in cell bodies and axon terminals of all Tv neurons. At this concentration of ETH1, calcium dynamics typically are characterized by transient, spike-shaped fluctuations superimposed upon a slow upward shift of the baseline, beginning ~8 min after exposure to the peptide. This response lasts ~10-15 min, after which weaker spike-like fluctuations continue without baseline changes until the end of recordings (~40 min). It is estimated that a concentration of 600 nM ETH1 results from a dose of w0.4 pmol of the peptide in vivo. Thus the major calcium response of Tv neurons coincides with the early phase of pre-ecdysis, and weaker activity persists through ecdysis and postecdysis. In contrast, ETH2 alone (600 nM) generates Ca2+ responses after a longer delay comparable to one following exposure to 60 nM ETH1. The longer delay of Ca2+ responses after ETH2 fits with the observations of in vivo behavior, where ETH2 is a less potent agonist than ETH1. The cocktail of ETH1 and ETH2 (600 nM each) evokes Ca2+ dynamics after a delay similar to that induced by ETH1 alone. Overall, [Ca2+]i dynamics observed in Tv neurons are synchronized. In many preparations, Tv neurons from the same neuromere appear to be strongly coupled, given that they produce precisely synchronized Ca2+ dynamics. Transient Ca2+ signals are obvious in the terminal processes of Tv neurons in NHO, the release sites of FMRFamides (Kim, 2006a).

Lower concentrations of ETH1 elicit calcium dynamics after a somewhat longer delay. Interestingly, calcium dynamics are obvious first in neurohemal endings of the NHO, followed by fluctuations in cell bodies. This was particularly evident at 60 nM ETH1, where a robust calcium response in the NHO was accompanied by only a weak response in the Tv2 cell body. No calcium responses are observed in Tvs exposed to 6 nM ETH1 (Kim, 2006a).

EH neurons reach peak activity at ecdysis: VM neurons producing eclosion hormone (EH) have been implicated as primary ETH targets during fly and moth ecdysis. Expression of ETHR-A was demonstrated in VM neurons, confirming that they are primary ETH targets. To determine whether ETH elicits activity in EH neurons, transgenic flies were prepared doubly homozygous for EHup-GAL4 and UASGCaMP, that show GCaMP fluorescence only in these cells (Kim, 2006a).

EH neurons are highly sensitive to ETH1, exhibiting robust [Ca2+]i dynamics upon exposure to concentrations as low as 6 nM. No detectable fluorescence responses are observed after exposure to 0.6 nM ETH1 over a period of 50-60 min. The latency to Ca2+ responses is inversely proportional to the concentration of ETH1; higher ETH1 concentrations evoke faster responses. The cocktail of ETH1 and ETH2 (600 nM each) elicited Ca2+ responses after a ~10-15 min delay (Kim, 2006a).

Close examination of these ETH-evoked fluorescence responses reveals two components distinguished by slow and fast kinetics. The slow component is characterized by a gradual increase in baseline levels of Ca2+ followed by a decrease over 20-30 min, whereas the fast component is composed of transient, spike-like activity. Fast components have durations ranging from 5-20 s. Peak DF/F responses are quite variable, even among a group of neurons exposed to the same ETH1 concentration. No significant concentration dependence could be detected in peak response (Kim, 2006a).

Distinct subsets of neurons 27/704 are active during different phases of the ecdysis sequence: ETH-evoked Ca2+ signals of neurons 27/704 were examined in transgenic flies carrying CCAP-GAL4 and UAS-GCaMP. Use of the CCAP promoter to drive GCaMP expression resulted in a reporter pattern identical to that described previously. Upon exposure to 600 nM ETH1, distinct subsets of neurons 27/704 exhibited reproducible, stereotypic Ca2+ responses in terms of peak intensity, latency, and termination of Ca2+ dynamics. According to the magnitude of peak fluorescence intensity (peak DF/F), neurons 27/704 fall into three major groups: strong responders, weak responders, and nonresponders. The strong-responder group includes neurons 27/704 in AN1-4 (CCAP/MIPs/bursicon), AN8,9 (CCAP/ MIPs), and TN3 (CCAP). Weak responders are neurons 27/704 in SN2-3, TN1-2, and AN7 producing CCAP only. Neurons in the brain, SN1, and AN5,6 showed no reproducible Ca2+ dynamics in response to 600 nM ETH1 (Kim, 2006a).

In response to ETH1, neurons 27/704 in TN3 and AN8,9 become active within 10-15 min, whereas neurons 27/704 in AN1-4 are activated after a 15-25 min delay. Neurons in TN3 and AN8,9 are therefore activated just prior to ecdysis onset, indicating their possible roles in initiation and maintenance of ecdysis behavior. In addition, Ca2+ dynamics observed in AN8,9 neurons terminated early in postecdysis, supporting this interpretation. In contrast, Ca2+ dynamics of neurons in AN1-4 begin during ecdysis and increase in intensity during the entire postecdysis period, suggesting their roles in these events. The cocktail of ETH1 and ETH2 (600 nM each) evoked Ca2+ dynamics similar to those induced by ETH1 alone. Two groups of neurons 27/704 in abdominal neuromeres (AN1-4 versus AN8,9) exhibit differences in sensitivity to ETH and in their patterns of [Ca2+]i dynamics. It was found that 6-60 nM ETH1 activates neurons in AN1-4 (n = 4), whereas higher concentrations of ETH1 (R600 nM) are required to activate neurons in AN8,9. In addition, neurons in AN8,9 generate transient (1-2 min) Ca2+ spikes over a 15-20 min period after ETH1 activation, whereas neurons in AN1-4 generally produce slower, more persistent Ca2+ dynamics. These differences among subgroups of neurons 27/704 suggest their different functional roles during the ecdysis sequence (Kim, 2006a).

Targeted ablations of apecific ETHR neurons have behavioral consequences: To evaluate behavioral roles of specific ETHR neurons, phenotypes of the pupal ecdysis sequence were investigated in transgenic flies bearing targeted ablations of ETHR neurons, including Tv FMRFamide neurons, EH neurons, and CCAP neurons (27/704 homologs). In control flies carrying UAS-reaper and UAS-GCaMP, but lacking the GAL4 driver, pupal ecdysis was executed as in wild-type flies: pre-ecdysis (0-10 min), ecdysis (10-23 min), and postecdysis (23-100 min) (Figure 7). Given that puparium-intact animals were used, the duration of ecdysis behavior may have been overestimated. Transgenic flies bearing targeted ablations of Tv FMRFamide neurons (FMRF-KO) were generated by crossing females doubly homozygous for FMRFa-GAL4, UAS-GCaMP with homozygous UAS-reaper males. Pupal ecdysis of FMRFa-KO flies is very similar to that of control flies. Because FMRFa-GAL4 drives expression of GAL4 only in three pairs of thoracic Tv neurons and one pair of unidentified neurons in SN, FMRFa-KO flies lost only Tv neurons and not other FMRFamide neurons in the CNS. FMRFa-KO flies complete pupal ecdysis without any detectable abnormality, except that pre-ecdysis contractions appear weaker than in control flies. Pupal ecdysis of VM neuron knockout flies (EH-KO) was then examined. Behavioral analysis showed that, although they complete pupal ecdysis without any severe defects or lethality, ecdysis onset is delayed w4 min. As a result of this delay, EH-KO flies show longer pre-ecdysis than control flies. Additional parameters governing pre-ecdysis, ecdysis, and postecdysis are indistinguishable between EH-KO and control flies (Kim, 2006a).

Finally, CCAP-KO flies were generated in order to examine the functional roles of neurons 27/704 (CCAP neurons) in pupal ecdysis. As expected, CCAP-KO flies failed to initiate ecdysis contractions and could not complete head eversion. Instead, they show prolonged pre-ecdysis contractions for ~25 min, followed by weak random contractions of the abdomen (different from ecdysis and postecdysis contractions of control flies) for the next 50 min (Kim, 2006a).

Conclusions: This study has described orchestration of an innate behavior, the Drosophila pupal ecdysis sequence, by the endocrine peptide ETH. ETH release coincides with onset of behavior, and injection of ETH triggers the complete behavioral sequence, consistent with its role in ecdysis activation previously established in the moths Manduca sexta and Bombyx mori and in Drosophila larvae and adults. Absence of ETH causes lethal ecdysis deficiency, a phenotype that is rescued by ETH injection. ETH therefore functions as a 'command chemical' to orchestrate an innate behavior. Primary CNS targets of ETH was identified by using ETHR-specific in situ hybridization. ETHR-A occurs in multiple classes of peptidergic neurons producing EH, CCAP/MIPs/bursicon, FMRFamides, or kinin (Kim, 2006a).

Expression of ETHR-A was shown in VM neurons, which release EH. In response to ETH, VM neurons become active prior to ecdysis behavior and reach peak levels of activity during ecdysis. These results provide further support for a previously described positive-feedback signaling pathway between VM neurons and Inka cells. This feedback is thought to ensure depletion of ETH from Inka cells. These findings are striking because independent evidence indicates that homologous VM neurons in the moth Manduca are direct targets of ETH and that their secretory products regulate ecdysis behaviors downstream of ETH. For example, isolated EH neurons of Manduca respond to direct action of ETH with increased excitability and spike broadening. In response to ETH action, these neurons release EH, causing cGMP elevation and increased excitability in CCAP-containing neurons 27/704 of the thoracic and abdominal ganglia. CCAP and MIPs, cotransmitters produced by neurons 704, are implicated in eliciting ecdysis behavior (Kim, 2006a).

A homologous role for EH in activation of Drosophila 27/704 neurons has not been clearly demonstrated. For example, no cGMP elevation is observed in these neurons during the natural ecdysis sequence. This lack of cGMP elevation suggests that CCAP neurons are not directly targeted by EH in Drosophila. Nevertheless, EH-knockout flies exhibit a delay in ecdysis initiation, suggesting that EH may modulate excitability in 27/704 cells indirectly through release of additional factors within the CNS. It is therefore proposed that activation of EH neurons by ETH serves two purposes: (1) release of EH into the hemocoel functions as part of a positive-feedback pathway to ensure ETH depletion from Inka cells; (2) release of EH within the CNS synergizes direct ETH actions on different subsets of neurons 27/704 producing CCAP, MIPs, and bursicon, perhaps indirectly through release of downstream signals within the CNS (Kim, 2006a).

Neurons 27/704 expressing ETHR-A respond to ETH with unique patterns of Ca2+ dynamics. These neurons are subdivided by pattern of transmitter expression: CCAP/MIPs/bursicon in AN1-4; CCAP/MIPs in AN8,9; and CCAP in SN1-3, TN1-3, and AN5-7. Temporal patterns of Ca2+ dynamics were determined in each neuronal subset relevant to the behaviors observed. On the basis of these temporal patterns, it is proposed that direct action of ETH on neurons 27/704 in TN3 and in AN8,9 induces initiation and execution of ecdysis contractions and head eversion. In support of this, it is shown that ablation of CCAP neurons abolishes ecdysis contractions and head eversion. Parallel study in Manduca showed that neurons 704 expressing ETHR-A and their peptide cotransmitters, CCAP and MIPs, are implicated in control of the ecdysis motor pattern, supporting the homologous function of 27/704 neurons in Drosophila. Neurons 27/704 in AN1-4 produce CCAP, MIPs, and bursicon, and therefore a cocktail of these peptides is likely released within the CNS and into the hemolymph during postecdysis. It is suggested that centrally released peptides control postecdysis movements, whereas blood-borne CCAP/MIPs regulate heart beat and blood pressure for cuticle expansion and bursicon controls sclerotization of expanded new cuticle. Bursicon was recently identified as a heterodimeric peptide hormone regulating cuticle plasticization, sclerotization, and melanization (Kim, 2006a).

The Drosophila FMRFamide gene (FMRFa) encodes multiple FMRFamide-related neuropeptides, which are expressed in many different cell types, including neuroendocrine cells, interneurons, and perhaps motoneurons. Among these diverse FMRFamide-producing neurons, ETHR-A expression is confined to three pairs of thoracic neurosecretory neurons, Tv1-3. Results of the present study show that the Tv neurons are activated early in pre-ecdysis and that they remain active during ecdysis and postecdysis. However, FMRFa-KO flies show no differences in timing of the ecdysis behavioral sequence. Because FMRFamides enhance twitch tension of larval body-wall muscles through synaptic modulation at the neuromuscular junction, blood-borne FMRFamides released from Tv neurons likely facilitates pre-ecdysis, ecdysis, and postecdysis contractions. Thus the role of Tv neurons as primary ETH targets may be enhancement of muscle contraction during the behaviors. Further work to substantiate this is in progress (Kim, 2006a).

Expression of ETHR-A occurs in in kinin neurons of abdominal neuromeres of Drosophila. Drosophila kinin is known to be involved in water balance, but its central functions have not been described or considered. Expression of ETHR-A in kinin neurons appears to be a conserved mechanism in fly and moth; the Manduca ETHR-A is expressed in abdominal neurosecretory cells (L3,4), which produce kinins and diuretic hormones (DHs). It was further found that the isolated Manduca CNS generates the fictive pre-ecdysis motor pattern upon exposure to a cocktail of kinin and DHs. These findings suggest that ETH activates L3,4 neurons in Manduca to release kinins and DHs centrally, which initiate and execute pre-ecdysis. On the basis of the conservation between Drosophila and Manduca in spatial expression pattern of ETHR, it is proposed that ETH initiates pre-ecdysis behavior indirectly via central release of kinin in Drosophila (Kim, 2006a).

In Drosophila, pupal ecdysis is accomplished by sequential recruitment of three major behavioral units: pre-ecdysis (0-10 min), ecdysis (10-15 min), and postecdysis (15-100 min). Each behavioral unit is programmed in the CNS and sequentially activated by direct actions of ETH, which is synthesized and released from peripheral endocrine Inka cells. Around 4-5 min before pre-ecdysis onset, a sizeable portion (~50%) of Inka cells initiates secretion of ETH into the hemolymph, whereas the remaining portion completes secretion after onset of pre-ecdysis. Appearance of ETH in the hemolymph activates ETHR-A in neurons expressing neuropeptides including kinin, FMRFamides (Tv1-3), EH, or CCAP, MIPs, and bursicon, but they are not released until descending inhibition is removed at key times during the ecdysis sequence. Upon activation of ETHR, the central release of kinin initiates pre-ecdysis contractions, whereas Tv neurons secrete FMRFamides to enhance neuromuscular transmission. ETH activates neurons producing EH, CCAP, CCAP/MIPs, and CCAP/MIPs/bursicon at different times. EH cells in the brain and neurons producing CCAP in TN3 and CCAP/MIPs in AN8,9 become active ~10-13 min after pre-ecdysis initiation. EH participates in timing the activation of ecdysis neurons, whereas CCAP and MIPs from TN3 and AN8,9 control initiation and execution of the ecdysis motor program. At the end of ecdysis (25 min after pre-ecdysis onset), neurons in AN1-4 secrete a cocktail of CCAP, MIPs, and bursicon, which likely regulate postecdysis contractions and processes associated with cuticle expansion, hardening, and tanning (Kim, 2006a).

This study has mapped central ETH receptor neurons, and discovered that they comprise multiple peptidergic ensembles, which are recruited sequentially to generate each phase of the ecdysis sequence. Ensemble-specific knockout analysis supports this interpretation. Each step of the ecdysis sequence (pre-ecdysis, ecdysis, postecdysis) is driven by a central pattern generator (CPG) within the CNS in the absence of sensory input. It is known that amines and peptides can modulate and reconfigure neuronal circuits comprising CPGs so as to elicit a variety of motor patterns. It seems likely that the multiple peptidergic ensembles described in this study as targets for ETH may be involved in configuring and activating CPGs underlying each step of the ecdysis sequence (Kim, 2006a).

Processes in the brain that govern behaviors over longer time frames such as sleep, mood, sexual activities, and even learning and memory could be associated with coordinated release of neuromodulators such as peptides. Further work on activation of central peptidergic ensembles in the CNS may shed light on mechanisms underlying release of a variety of behaviors (Kim, 2006a).

Protein Interactions

G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous 'orphan' GPCRs have become available for functional analysis. This study analyzes two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins (FXPRXamide), Cap2b-like peptides (FPRXamide), or ecdysis triggering hormones (PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (CCAP), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors (Park, 2002b).

Examination of the three Drosophila GPCRs homologous to the AVP receptor yielded serendipitous findings. CG6111, orthologous to the vasopressin receptor, is activated by CCAP and AKH. CG10698 and CG11325 are activated by corazonin and AKH, respectively. The EC50 values for receptors in the AVP group are consistently lower than those observed in the NMU PRXa group (Park, 2002b).

It is surprising that CG6111, an orthologous gene of AVP receptor, is activated by CCAP and AKH, but not by AVP. The presence of an insect vasopressin-like peptide was reported in locust, but searches of the Drosophila genome sequence to locate a candidate AVP-like peptide sequence have been unsuccessful. CCAP and AVP both are C-terminally amidated, disulfide bridged peptides, but share no significant sequence similarity. The current data set favors assignment of CG6111 as an authentic CCAP receptor because of ligand cross-reactivity within this group of GPCRs. It seems reasonable to have residual functional cross-activity within recent evolutionarily diverged GPCRs. Further work is needed to verify whether CG6111 is an authentic CCAP receptor or is a receptor for unidentified Drosophila AVP-like peptide cross-reacting to the CCAP (Park, 2002b).

CG10698 is activated by corazonin with an EC50 of 1 nM. Similarly, CG11325, previously cloned by its homology to GNRHR, is activated by AKH with an EC50 of 0.3 nM. These evolutionarily related GPCRs, activated by structurally similar signaling peptides, reveals a clear case of receptor-ligand coevolution (Park, 2002b).

CCAP, corazonin, and AKH have overlapping biological functions, and thus it is not unexpected that their receptors would fall into an evolutionarily related group. CCAP was initially identified by its cardioacceleratory action on the heart of the shore crab and in the tobacco hawkmoth, Manduca. The primary structure of this peptide appears to be strictly conserved across the arthropods. Additional functions of CCAP include myotropic actions, induction of AKH release in corpora cardiaca of locust, and induction of ecdysis behaviors. Corazonin is known for its cardioactive function in cockroach and pigment modulation in locust. AKH and related peptides, grouped with red pigment concentrating hormone of crustacea are cardioacceleratory and have metabolic functions such as lipid and carbohydrate mobilization (Park, 2002b).

The present findings favor a hypothesis that the PRXa motif is an evolutionarily conserved signature in both vertebrate NMU and insect PRXa peptides. Examination of potential cross-activity of NMU and insect PRXa peptides for their receptors may provide further support for the theory of ligand-receptor coevolution in the PRXa peptide-receptor group. Another clear case of ligand-receptor coevolution has been shown for recently diverging corazonin and AKH, and their receptors (Park, 2002b).

The ligand-activated GPCR responses described in this report provide an important first step in defining authentic physiological roles for these signaling peptides. These findings help to promote a subset of GPCRs from 'orphan' to 'putative' receptors, and provide a direction for further characterization of receptors for PRXa peptides, CCAP, corazonin, and AKH. Expanded studies in Drosophila and in other insects will help to validate these initial findings (Park, 2002b).

The multiple ligand sensitivity exhibited by certain GPCRs such as CG8795 and CG6111 raises the obvious question of physiological significance. One possible interpretation is that, in contrast to a one ligand-one receptor model, certain receptors may be involved in the transduction of multiple peptide signals, thus providing a pleiotropic model of functional regulation. This possibility deserves careful examination in more physiologically relevant bioassays, as well as in vivo (Park, 2002b).

DEVELOPMENTAL BIOLOGY

DIG-labeled antisense probes synthesized using the Ccap cDNA were used to determine the in situ pattern of expression of the gene corresponding to this cDNA in the CNS of 3rd instar larvae. The observed pattern of RNA expression matches that of the known patterns of Ccap-immunoreactivity [Ccap-IR; (Ewer, 1996)]. In Drosophila the Ccap peptide is consistently expressed in 2 pairs of neurons in the brain, 5 pairs in the subesophageal ganglion, 1-2 pairs in at least 8 ganglia of the ventral nervous system, as well as in 2 pairs of strongly immunoreactive descending axons, one lateral and one medial (see Ewer, 1996). Processing these tissues simultaneously for both RNA expression and immunoreactivity revealed that Ccap immunoreactivity and Ccap mRNA are always co-localized. This complete concordance between the two signals, coupled with the sequence information of the Ccap gene, demonstrate that the cloned sequence encodes the Ccap peptide (Park, 2003).

In Drosophila, a circadian clock controls the timing of adult emergence, with most adults eclosing between subjective dawn and late subjective morning. Although much is known about the circadian clock mechanism, comparatively little is known about how the clock regulates the expression of overt rhythmicity. The co-localization of Lark and Ccap suggests that Ccap neurons could mediate the circadian control of ecdysis, independent of its possible role in the execution of the behavior itself. To determine whether the clock directly regulates the Ccap cells, the relative locations of the clock and Ccap neuronal populations in larval and pharate adult brains were examined. This was accomplished by examining Ccap immunoreactivity in brains expressing green fluorescent protein (GFP) in the clock cell population. Using a timeless-GAL4 driver it was observed that projections from the Tim-containing DN2 neurons overlap with Ccap-immunoreactive synaptic endings in the dorsal aspect of the larval and pharate adult brains. Interestingly, DN2 neurons are postulated to be targets of Pigment dispersing factor (PDF)-containing small ventral lateral neurons (LN_v), and they have been implicated in the circadian control of locomotor activity. In a separate experiment using a pdf-GAL4 driver, overlap has been demonstrated between the processes of Ccap neurons and those of tritocerebral Pdf neurons. The latter population arises post-embryonically at the mid-pupal stage, and it has been suggested that it might be involved with the circadian control of adult eclosion (Park, 2003).

Analysis of the Drosophila lark gene indicates that it encodes an RNA-binding protein that functions as a regulatory element of the circadian clock output pathway controlling adult eclosion. The Lark RNA-binding protein oscillates in abundance during the circadian cycle; importantly, the phasing of the Lark rhythm is consistent with gene-dosage studies, which indicate that the protein behaves as a repressor molecule. The Lark protein rhythm persists in constant conditions (continuous darkness and constant temperature) and is eliminated by period gene null mutations, confirming that it is under clock control and suggesting that it acts as an output mechanism that mediates the temporal regulation of adult eclosion. Lark protein oscillates in abundance within a defined group of neuropeptide (CCAP) -containing neurons of the ventral nervous system (VNS), which in other insects are thought to comprise cellular elements of the clock output pathway regulating eclosion (McNeil, 1998).

During insect metamorphosis, neuronal networks undergo extensive remodeling by restructuring their connectivity and recruiting newborn neurons from postembryonic lineages. The neuronal network that directs the essential behavior, ecdysis, generates a distinct behavioral sequence at each developmental transition. Larval ecdysis replaces the cuticle between larval stages, and pupal ecdysis externalizes and expands the head and appendages to their adult position. However, the network changes that support these differences are unknown. Crustacean cardioactive peptide (CCAP) neurons and the peptide hormones they secrete are critical for ecdysis; their targeted ablation alters larval ecdysis progression and results in a failure of pupal ecdysis. This study demonstrates that the CCAP neuron network is remodeled immediately before pupal ecdysis by the emergence of 12 late CCAP neurons. All 12 are CCAP efferents that exit the central nervous system. Importantly, these late CCAP neurons were found to be entirely sufficient for wild-type pupal ecdysis, even after targeted ablation of all other 42 CCAP neurons. Evidence indicates that late CCAP neurons are derived from early, likely embryonic, lineages. However, they do not differentiate to express their peptide hormone battery, nor do they project an axon via lateral nerve trunks until pupariation, both of which are believed to be critical for the function of CCAP efferent neurons in ecdysis. Further analysis implicated ecdysone signaling via ecdysone receptors A/B1 and the nuclear receptor ftz-f1 as the differentiation trigger. These results demonstrate the utility of temporally tuned neuronal differentiation as a hard-wired developmental mechanism to remodel a neuronal network to generate a scheduled change in behavior (Veverytsa, 2012; full text of article).

Specification of neuronal subtypes by different levels of Hunchback

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. This study analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. Both were found to be specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. The expression of Hunchback in the neuroblast increases over time, and evidence is provided that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates (Moris-Sanz, 2014).

This study analyzed how CCAP-expressing neurons are specified. Evidence was obtained that both the the efferent subset of CCAP neurons (CCAP-ENs) and interneuron subset (CCAP-INs) of all embryonic segments are generated by NB3-5. The results also indicate that CCAP neurons are generated in the Hb temporal window, are not sibling cells and that the CCAP-ENs are generated first followed by the CCAP-INs. Although the Hb temporal window in NB3-5 generates two GMCs that can be distinguished by the expression of Pdm in GMC1, Pdm does not seem to play any role in the specification of these neurons, as no phenotype was observed in pdm mutants (Moris-Sanz, 2014).

These findings raised the question of how these two neuronal fates are generated, and the results that are presented in this study suggest that different levels of Hb expression specify them. The evidence for this is as follows. First, Hb expression in NB3-5 increases over time from stage 9 to early stage 11, then its expression quickly fades, coinciding with the reported expression of Svp, which is known to close the Hb temporal window. During this time window, NB3-5 divides twice and generates four neurons. Second, overexpression of high levels of Hb using a pan-NB driver extends the IN fate. Third, in an hb hypomorphic condition CCAP-INs are lost or converted into ENs, as monitored by the expression of Dac and the presence of axons that exit the ganglion (Moris-Sanz, 2014).

This mechanism for generating distinct neuronal fates is different from that proposed for subdividing the Cas temporal window in NB5-6, which involves two sequential feed-forward loops and several genes to define the fates of four cells (Ap1-4) that are sequentially generated and form the Apterous (Ap) cluster of neurons. However, the mechanism that was proposed is very similar to the role that the grh gene plays in the Ap cluster, since Grh expression increases gradually over time from Ap1 to Ap4, and overexpression of Grh converts all four Ap neurons into Ap4 (Moris-Sanz, 2014).

In addition to the different levels of Hb expression observed in NB3-5, it was found that CCAP-ENs and CCAP-INs express low and high levels of Hb, respectively, and overexpression of Hb in postmitotic cells convert the ENs into INs. These observations raise the question of how a high level of Hb expression in the NB leads to a high level of expression in the neuron. A recent analysis of the hb regulatory region revealed a specific postmitotic enhancer, so it would be tempting to propose that this enhancer is only activated in neurons that are generated by a NB expressing a high level of Hb. However, no expression of this enhancer was detected in any of the CCAP neurons, and overexpression of Hb in the NB did not lead to activation of the enhancer in neurons. Therefore, further work is needed to identify the mechanism by which only a subset of the neurons generated in the Hb temporal window expresses a high level of Hb and how this is translated into different neuronal fates (Moris-Sanz, 2014).

CCAP-INs express a high level of Hb and do not express Dac, and upon Hb overexpression the expression of Dac is lost in many, although not all, cells. This could place dac as a direct target of Hb. Analysis of dac cis-regulatory domains indicates the presence of a 5.8 kb domain in the first intron that, when placed in a Gal4 vector, was sufficient to drive GFP expression in vivo in many neurons of late embryos . A preliminary analysis of the sequence of this domain suggests the presence of conserved regions and putative Hb binding sites. Further analysis will be required to confirm the presence and elucidate the function of such sequences (Moris-Sanz, 2014).

Ikaros (or Ikzf1), a mouse ortholog of Hb, is expressed in all early retinal progenitor cells (RPCs) of the developing retina. Its expression in RPCs is necessary and sufficient to confer the competence to generate early-born neurons. These and other observations suggest that, as in the Drosophila CNS, cell-intrinsic mechanisms act in the RPC to control temporal competence. Ikaros is expressed in the early RPCs that give rise to several cell types, namely horizontal, amacrine and gangion cells; however, it is unclear whether distinct levels of Ikaros expression are responsible for the production of these different cell types (Moris-Sanz, 2014).

In the early embryo, different concentrations of Hb seem to elicit different cellular responses. At low concentrations, Hb monomers function as activators, whereas at high concentrations they form dimers that either repress transcription or block activation. Analysis of the Hb protein has led to the identification of two conserved domains: a DNA-binding domain and a dimerization domain. More recently, it has been shown that, in CNS development, Hb repressor function is required to maintain early NB competence and to specify early-born neuronal identity. These results are compatible with the evidence presented in this study that it is the absolute level of Hb in a NB that determines whether it is expressed in the postmitotic progeny and so specifies the different neuronal subtypes (Moris-Sanz, 2014).

Role of the neuropeptide CCAP in Drosophila cardiac function

The heartbeat of adult Drosophila displays two cardiac phases, the anterograde and retrograde beat, which occur in cyclic alternation. Previous work demonstrated that the abdominal heart becomes segmentally innervated during metamorphosis by peripheral neurons that express crustacean cardioactive peptide (CCAP). CCAP has a cardioacceleratory effect when it is applied in vitro. The role of CCAP in adult cardiac function was studied in intact adult flies using targeted cell ablation and RNA interference (RNAi). Optical detection of heart activity showed that targeted ablation of CCAP neurons selectively altered the anterograde beat, without apparently altering the cyclic cardiac reversal. Normal development of the abdominal heart and of the remainder of cardiac innervation in flies lacking CCAP neurons was confirmed by immunocytochemistry. Thus, in addition to its important role in ecdysis behavior (the behavior used by insects to shed the remains of the old cuticle at the end of the molt), CCAP may control the level of activity of the anterograde cardiac pacemaker in the adult fly. Expression of double stranded CCAP RNA in the CCAP neurons (targeted CCAP RNAi) causes a significant reduction in CCAP expression. However, this reduction is not sufficient to compromise CCAP's function in ecdysis behavior and heartbeat regulation (Dulcis, 2005).

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).

Effects of Ccap neuron cellular knockout

To produce animals lacking Ccap neurons, expression of the cell death gene reaper (rpr) was driven in Ccap neurons, using the Ccap-GAL4 transgenic strain. A similar approach has been successfully used to study the function of other Drosophila neuropeptides and hormones. To investigate the consequences of loss of Ccap neurons on larval ecdysis, Ccap neurons had to be absent, at the latest, prior to the last larval ecdysis (from 2nd to 3rd larval instar). The targeted expression of rpr using the Ccap-GAL4 driver was shown to produce late 2nd instar larvae that are probably entirely devoid of Ccap function. In certain experiments that examined post-larval ecdyses, animals were transferred to 20°C after collection as first instar larvae and raised at this temperature until pupation or eclosion. At this lower temperature the vast majority of the CNSs was also mostly devoid of Ccap neurons by the end of the 3rd instar (at wandering). Thus, of 22 CNSs examined at this time, 15 showed no Ccap- or ß-gal-immunoreactive neurons or processes, while four, two and one CNSs had one, two and four weakly stained neurons, respectively, and none of these CNSs had visible immunoreactive processes. When the CNS of animals raised using the eclosion rhythm paradigm (25°C to 18°C) was processed for Ccap- and ß-gal-IR immediately after adult eclosion, 25 of 28 CNSs showed no immunoreactivity, while two and one CNSs had one and two weakly staining neurons, respectively, lacking visible processes (Park, 2003).

The role of Ccap in larval ecdysis

In the moth Manduca sexta, addition of CCAP to an isolated larval abdominal CNS turns on the ecdysis motor program (Gammie, 1997b; Zitnan, 2000). This, and other evidence (reviewed by Ewer, 2002), strongly implicates the CCAP neuropeptide in the control of ecdysis behavior in this moth. In the CNS of Drosophila larvae, Ccap-IR decreases shortly before the onset of larval ecdysis, suggesting that Ccap is similarly important for the control of ecdysis in this insect (Park, 2003).

In Drosophila, there are two larval intermolts, the intermolt between first and send larval instars, and the intermolt between second and third instars. To investigate directly the role of Ccap in larval ecdysis, animals lacking the Ccap neuronal population were characterized. Surprisingly, it was found that genetic ablation of the Ccap neurons is not lethal during the larval stages. Indeed, the survival rate of Ccap-KO from 1st instar to the end of the 3rd (last) instar is indistinguishable from that of the control population (97% vs. 95%, respectively). This indicates that Ccap is not essential for viability during (at least) the latter part of the 2nd larval intermolt period and the entire 3rd larval instar. Most significantly, animals lacking Ccap neurons were able to shed their old cuticle at the end of molt to the 3rd instar. Independent studies show that animals homozygous for small chromosomal deletions including Ccap (and 14 other genes) survive until the 3rd instar. Thus, survival of Ccap-KO larvae until this stage is not due to persisting (but immunohistochemically undetectable) Ccap peptide (Park, 2003).

To determine whether ecdysis behavior is normal in KO animals, the sequence and timing of ecdysis behavior from the 2nd to the 3rd instar was examined. The earliest obvious marker for the impending ecdysis is the appearance of pigmentation in the mouth plates of the future 3rd instar (double mouth plates stage; DMP), which occurs about 30 minutes before ecdysis. Approximately 16 minutes after the DMP stage, air enters the new trachea; this is followed shortly by the onset of the preparatory behavior called pre-ecdysis ( Park, 2002a). Approximately 15 minutes after air filling, pre-ecdysis stops and the animal executes a characteristic 'biting' behavior during which it appears to be attempting to tear the anterior region of the old cuticle. This period is then followed by the onset of ecdysis proper, which is characterized by vigorous peristaltic waves sweeping along the animal in a posterior-to-anterior direction. Typically after three to four waves, the anterior cuticle breaks, freeing the 3rd instar of its 2nd instar cuticle. After a period of a few minutes the animal resumes feeding and locomotory behavior (Park, 2003).

Although the Ccap-KO larvae are clearly able to initiate ecdysis behavior and use this behavior to free themselves from the 2nd instar cuticle, there are subtle but significant differences between the behavior of Ccap-KO and control animals. The duration of events up to the onset of pre-ecdysis is indistinguishable for these two groups of larvae. The first notable difference between Ccap-KOs and control larvae is a modest but significant lengthening of pre-ecdysis behavior, although pre-ecdysis behavior itself appears normal (Park, 2003).

Additionally, the subsequent events that lead to cuticle shedding takes approx. three times longer in Ccap-KO larvae than in controls. Both the biting period, which occurs between the end of pre-ecdysis and ecdysis onset, and the duration of ecdysis itself, are significantly extended in Ccap-KO animals. Interestingly, Ccap-KO larvae exhibit anterior to posterior peristaltic waves interspersed with the typically occurring posterior to anterior waves, a behavior never observed in control animals. Because the waves moving in the anterior to posterior direction do not aid in breaking the old cuticle, the time to successful shedding of the old cuticle is lengthened (Park, 2003).

These results reveal that the ablation of Ccap neurons is associated with defects that are strictly confined to the execution of ecdysis itself. Thus, while the entire duration of the period between DMP and ecdysis is increased by only 14%, from the normal 31.9±1.0 minutes to 36.3±0.9 minutes, the timing and organization of ecdysis behavior itself is quite severely disrupted in the KO animals (Park, 2003).

The role of Ccap in pupal ecdysis

In higher Diptera such as Drosophila , pupal ecdysis (pupation) corresponds to the behavior referred to as 'head eversion'. During head eversion, the brain, which in the larva is located behind the mouthparts, is pushed anteriorly to a position in front of the thorax and the mouthparts. At the same time, the appendages, which were formed from the eversion of the imaginal discs at pupariation (the onset of the larval-pupal transition), are extended to attain their final size and shape (Park, 2003).

In contrast to the situation observed in the larva, most Ccap-KO animals die during the pupal stage. Furthermore, the appearance of KO animals at the end of pupation and of metamorphosis suggests that the primary cause of their death is a specific failure of pupal ecdysis. Indeed, in KO pupae and pharate adults, the head is located much more to the anterior than normal or is only partially everted; the larval tracheae have not been completely shed; and the appendages have not been properly extended, resulting in a pharate adult that has abnormally short wings and legs (Park, 2003).

In order to determine the bases for these defects, the timecourse of pupal ecdysis was examined in KO animals. In Drosophila , pupation occurs approx. 12 hours after pupariation. Pupal ecdysis is preceded by a preparatory behavior (termed here pre-ecdysis by analogy to the corresponding larval behavior), during which the posterior part of the animal rhythmically retracts from the puparium. About 9-10 minutes after the onset of pre-ecdysis, a short succession of peristaltic waves sweeps from the posterior to the anterior of the animal and causes the eversion of the head, the shedding of the larval tracheae, and a rapid extension of the appendages. Head eversion is followed by a long post-ecdysis period of several hours during which regular contractions, primarily of the abdomen, occur; this presumably aids in giving the insect its final form (Park, 2003).

Ccap-KO animals initiate normal pre-ecdysis behavior (for instance the frequency of abdominal 'sweeps' is the same as in controls). However, this behavior lasts significantly longer than in controls and is not followed by head eversion. Instead, abdominal pre-ecdysis movements eventually cease during a final retraction and are followed by a period that resembles the postecdysis period seen in the control (but which is significantly shorter in Ccap-KO animals (Park, 2003).

Although the KO pre-pupae all lack Ccap neurons, the morphological phenotype seen at the end of metamorphosis is somewhat variable, with, for instance, a variable amount of the adult head visible at the end of adult development. However, all flies show shortened appendages, and all animals whose pupation behavior was observed in detail show no pupal ecdysis. The basis for this variable phenotype is currently unknown (Park, 2003).

If head eversion is stimulated by Ccap, the neuropeptide should be released at this time. Indeed, in wild type animals, a substantial decrease in Ccap-IR is detected following pupation in descending Ccap immunopositive axons. The slight increase in the number of Ccap-immunoreactive varicosities that is apparent at the start of pre-ecdysis is a reflection of a subtle fragmentation in the pattern of Ccap-IR that is seen at this time, and may be the first sign that Ccap has started to be released. The ETH peptides are known to be essential for larval pre-ecdysis in Drosophila (Park, 2002a), and the drop in ETH-IR that is observed at the onset of pupal pre-ecdysis suggests that these peptides may also control this behavior at pupation (Park, 2003).

The role of Ccap in adult ecdysis (eclosion)

The KO animals that formed relatively normal heads at the end of metamorphosis are usually able to exit from the pupal case. A careful examination of eclosion has shown that the developmental and behavioral events that take place at this time occurs in the correct sequence in Ccap-KO animals, although some quantitative differences in the duration or number of events are observed. Thus, while tracheal filling occurs before the start of the eclosion behaviors, in Ccap-KO animals it takes longer than in controls. However, the ptilinum, which is used to rupture the anterior of the pupal case, is deployed normally at the expected time. Finally, and most significantly, the bouts of rapid anteriorly directed peristalses of ecdysis proper occurred in the KO animals. Interestingly, however, these bouts are relatively ineffective at propelling the animal forward. This is not due to a difference in the characteristics of the bouts themselves, which careful cinematographic analyses shows are very similar to those of control animals. Instead, this failure occurred because the abdomen of KO animals is not distended at this time, severely reducing the traction exerted by the body on the inner walls of the pupal case: this is needed in order for the abdominal peristalses to cause the rapid net forward movement that is seen in control animals. Although most KO animals (nine out of 10 examined) eventually succeeded in eclosing, extrication took much longer than normal. Thus, unlike larval and pupal ecdysis, the actual ecdysis motor program of the adult appears to be relatively normal in KO animals (however, the frequency of peristalses is lower than in controls, even when the two groups are compared during the first minute after the onset of eclosion, which corresponds approximately to the duration of eclosion in controls. Therefore, the most dramatic deficiency of Ccap-KO animals at eclosion appears to be due to the absence of a function required to expand the body rather than to a failure in the adult ecdysis motor program itself (Park, 2003).

The adult phenotype

The phenotypes of adult Ccap-KO flies suggest that Ccap neurons play some role in post-eclosion events. KO adults do not inflate their wings, and their cuticle appears to remain soft and untanned, as evidenced by the dimpling that is observed on the dorsal thorax at sites of thoracic muscle insertion. The defect in wing expansion may be due, in part, to the failure in wing extension at the time of pupation (Park, 2003). The tanning defect of the KO flies may occur because a subset of the Ccap neurons expresses the gene encoding the tanning hormone, bursicon (E. Dewey and H. W. Honegger, personal communication to Park, 2003).

In another experiment, the Ccap-GAL4 driver was used to overexpress a temperature-sensitive form of shibire (shi; the fly dynamin homolog) in the CCAP cell population (using a UAS-shi^ts transgene). When reared at 29°C, progeny carrying both the Ccap-GAL4 and UAS-shi^ts transgenes exhibited defects in wing expansion ~80%-100% of the populations), whereas control progeny (with only one transgene) have normal wings. At 25°C, both types of progeny have normal wings, indicative of a temperature-sensitive effect (Park, 2003).

Eclosion rhythms in the absence of Ccap neurons

To examine circadian rhythms of eclosion, Ccap-KO and control animals were reared under conditions that produced the maximal number of pharate adults, and then adult emergence was scored at two-hour intervals over the course of several days, both under a light:dark cycle (LD 12:12) and in constant darkness (DD). In three separate experiments using one Ccap-GAL4 line and in two separate experiments using a second independent transgenic line, a clear rhythmicity was observed under both LD and DD conditions, with most of the animals eclosing in the dawn-early morning (or subjective dawn-early morning) interval. Nevertheless, there were differences between the eclosion profiles of KO and control populations. Most notably, the temporal gate of eclosion was lengthened in KO animals, with significant emergence occurring in the late night/predawn period. Coupled with this wider eclosion 'gate', a significant diminution was observed in the amplitude of the eclosion burst that occurs immediately following lights-on, which in control populations constitutes approximately 40% of the flies that emerge on any given day. No consistent difference in the peak time of eclosion was observed between KO and control populations in LD or DD conditions (Park, 2003).

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).

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).

EFFECTS OF MUTATION

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

DIG-labeled antisense probes synthesized using the Ccap cDNA were used to determine the in situ pattern of expression of the gene corresponding to this cDNA in the CNS of 3rd instar larvae. The observed pattern of RNA expression matches that of the known patterns of Ccap-immunoreactivity [Ccap-IR; (Ewer, 1996)]. In Drosophila the Ccap peptide is consistently expressed in 2 pairs of neurons in the brain, 5 pairs in the subesophageal ganglion, 1-2 pairs in at least 8 ganglia of the ventral nervous system, as well as in 2 pairs of strongly immunoreactive descending axons, one lateral and one medial (see Ewer, 1996). Processing these tissues simultaneously for both RNA expression and immunoreactivity revealed that Ccap immunoreactivity and Ccap mRNA are always co-localized. This complete concordance between the two signals, coupled with the sequence information of the Ccap gene, demonstrate that the cloned sequence encodes the Ccap peptide (Park, 2003).

In Drosophila, a circadian clock controls the timing of adult emergence, with most adults eclosing between subjective dawn and late subjective morning. Although much is known about the circadian clock mechanism, comparatively little is known about how the clock regulates the expression of overt rhythmicity. The co-localization of Lark and Ccap suggests that Ccap neurons could mediate the circadian control of ecdysis, independent of its possible role in the execution of the behavior itself. To determine whether the clock directly regulates the Ccap cells, the relative locations of the clock and Ccap neuronal populations in larval and pharate adult brains were examined. This was accomplished by examining Ccap immunoreactivity in brains expressing green fluorescent protein (GFP) in the clock cell population. Using a timeless-GAL4 driver it was observed that projections from the Tim-containing DN2 neurons overlap with Ccap-immunoreactive synaptic endings in the dorsal aspect of the larval and pharate adult brains. Interestingly, DN2 neurons are postulated to be targets of Pigment dispersing factor (PDF)-containing small ventral lateral neurons (LN_v), and they have been implicated in the circadian control of locomotor activity. In a separate experiment using a pdf-GAL4 driver, overlap has been demonstrated between the processes of Ccap neurons and those of tritocerebral Pdf neurons. The latter population arises post-embryonically at the mid-pupal stage, and it has been suggested that it might be involved with the circadian control of adult eclosion (Park, 2003).

Analysis of the Drosophila lark gene indicates that it encodes an RNA-binding protein that functions as a regulatory element of the circadian clock output pathway controlling adult eclosion. The Lark RNA-binding protein oscillates in abundance during the circadian cycle; importantly, the phasing of the Lark rhythm is consistent with gene-dosage studies, which indicate that the protein behaves as a repressor molecule. The Lark protein rhythm persists in constant conditions (continuous darkness and constant temperature) and is eliminated by period gene null mutations, confirming that it is under clock control and suggesting that it acts as an output mechanism that mediates the temporal regulation of adult eclosion. Lark protein oscillates in abundance within a defined group of neuropeptide (CCAP) -containing neurons of the ventral nervous system (VNS), which in other insects are thought to comprise cellular elements of the clock output pathway regulating eclosion (McNeil, 1998).

During insect metamorphosis, neuronal networks undergo extensive remodeling by restructuring their connectivity and recruiting newborn neurons from postembryonic lineages. The neuronal network that directs the essential behavior, ecdysis, generates a distinct behavioral sequence at each developmental transition. Larval ecdysis replaces the cuticle between larval stages, and pupal ecdysis externalizes and expands the head and appendages to their adult position. However, the network changes that support these differences are unknown. Crustacean cardioactive peptide (CCAP) neurons and the peptide hormones they secrete are critical for ecdysis; their targeted ablation alters larval ecdysis progression and results in a failure of pupal ecdysis. This study demonstrates that the CCAP neuron network is remodeled immediately before pupal ecdysis by the emergence of 12 late CCAP neurons. All 12 are CCAP efferents that exit the central nervous system. Importantly, these late CCAP neurons were found to be entirely sufficient for wild-type pupal ecdysis, even after targeted ablation of all other 42 CCAP neurons. Evidence indicates that late CCAP neurons are derived from early, likely embryonic, lineages. However, they do not differentiate to express their peptide hormone battery, nor do they project an axon via lateral nerve trunks until pupariation, both of which are believed to be critical for the function of CCAP efferent neurons in ecdysis. Further analysis implicated ecdysone signaling via ecdysone receptors A/B1 and the nuclear receptor ftz-f1 as the differentiation trigger. These results demonstrate the utility of temporally tuned neuronal differentiation as a hard-wired developmental mechanism to remodel a neuronal network to generate a scheduled change in behavior (Veverytsa, 2012; full text of article).

Specification of neuronal subtypes by different levels of Hunchback

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. This study analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. Both were found to be specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. The expression of Hunchback in the neuroblast increases over time, and evidence is provided that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates (Moris-Sanz, 2014).

This study analyzed how CCAP-expressing neurons are specified. Evidence was obtained that both the the efferent subset of CCAP neurons (CCAP-ENs) and interneuron subset (CCAP-INs) of all embryonic segments are generated by NB3-5. The results also indicate that CCAP neurons are generated in the Hb temporal window, are not sibling cells and that the CCAP-ENs are generated first followed by the CCAP-INs. Although the Hb temporal window in NB3-5 generates two GMCs that can be distinguished by the expression of Pdm in GMC1, Pdm does not seem to play any role in the specification of these neurons, as no phenotype was observed in pdm mutants (Moris-Sanz, 2014).

These findings raised the question of how these two neuronal fates are generated, and the results that are presented in this study suggest that different levels of Hb expression specify them. The evidence for this is as follows. First, Hb expression in NB3-5 increases over time from stage 9 to early stage 11, then its expression quickly fades, coinciding with the reported expression of Svp, which is known to close the Hb temporal window. During this time window, NB3-5 divides twice and generates four neurons. Second, overexpression of high levels of Hb using a pan-NB driver extends the IN fate. Third, in an hb hypomorphic condition CCAP-INs are lost or converted into ENs, as monitored by the expression of Dac and the presence of axons that exit the ganglion (Moris-Sanz, 2014).

This mechanism for generating distinct neuronal fates is different from that proposed for subdividing the Cas temporal window in NB5-6, which involves two sequential feed-forward loops and several genes to define the fates of four cells (Ap1-4) that are sequentially generated and form the Apterous (Ap) cluster of neurons. However, the mechanism that was proposed is very similar to the role that the grh gene plays in the Ap cluster, since Grh expression increases gradually over time from Ap1 to Ap4, and overexpression of Grh converts all four Ap neurons into Ap4 (Moris-Sanz, 2014).

In addition to the different levels of Hb expression observed in NB3-5, it was found that CCAP-ENs and CCAP-INs express low and high levels of Hb, respectively, and overexpression of Hb in postmitotic cells convert the ENs into INs. These observations raise the question of how a high level of Hb expression in the NB leads to a high level of expression in the neuron. A recent analysis of the hb regulatory region revealed a specific postmitotic enhancer, so it would be tempting to propose that this enhancer is only activated in neurons that are generated by a NB expressing a high level of Hb. However, no expression of this enhancer was detected in any of the CCAP neurons, and overexpression of Hb in the NB did not lead to activation of the enhancer in neurons. Therefore, further work is needed to identify the mechanism by which only a subset of the neurons generated in the Hb temporal window expresses a high level of Hb and how this is translated into different neuronal fates (Moris-Sanz, 2014).

CCAP-INs express a high level of Hb and do not express Dac, and upon Hb overexpression the expression of Dac is lost in many, although not all, cells. This could place dac as a direct target of Hb. Analysis of dac cis-regulatory domains indicates the presence of a 5.8 kb domain in the first intron that, when placed in a Gal4 vector, was sufficient to drive GFP expression in vivo in many neurons of late embryos . A preliminary analysis of the sequence of this domain suggests the presence of conserved regions and putative Hb binding sites. Further analysis will be required to confirm the presence and elucidate the function of such sequences (Moris-Sanz, 2014).

Ikaros (or Ikzf1), a mouse ortholog of Hb, is expressed in all early retinal progenitor cells (RPCs) of the developing retina. Its expression in RPCs is necessary and sufficient to confer the competence to generate early-born neurons. These and other observations suggest that, as in the Drosophila CNS, cell-intrinsic mechanisms act in the RPC to control temporal competence. Ikaros is expressed in the early RPCs that give rise to several cell types, namely horizontal, amacrine and gangion cells; however, it is unclear whether distinct levels of Ikaros expression are responsible for the production of these different cell types (Moris-Sanz, 2014).

In the early embryo, different concentrations of Hb seem to elicit different cellular responses. At low concentrations, Hb monomers function as activators, whereas at high concentrations they form dimers that either repress transcription or block activation. Analysis of the Hb protein has led to the identification of two conserved domains: a DNA-binding domain and a dimerization domain. More recently, it has been shown that, in CNS development, Hb repressor function is required to maintain early NB competence and to specify early-born neuronal identity. These results are compatible with the evidence presented in this study that it is the absolute level of Hb in a NB that determines whether it is expressed in the postmitotic progeny and so specifies the different neuronal subtypes (Moris-Sanz, 2014).

Role of the neuropeptide CCAP in Drosophila cardiac function

The heartbeat of adult Drosophila displays two cardiac phases, the anterograde and retrograde beat, which occur in cyclic alternation. Previous work demonstrated that the abdominal heart becomes segmentally innervated during metamorphosis by peripheral neurons that express crustacean cardioactive peptide (CCAP). CCAP has a cardioacceleratory effect when it is applied in vitro. The role of CCAP in adult cardiac function was studied in intact adult flies using targeted cell ablation and RNA interference (RNAi). Optical detection of heart activity showed that targeted ablation of CCAP neurons selectively altered the anterograde beat, without apparently altering the cyclic cardiac reversal. Normal development of the abdominal heart and of the remainder of cardiac innervation in flies lacking CCAP neurons was confirmed by immunocytochemistry. Thus, in addition to its important role in ecdysis behavior (the behavior used by insects to shed the remains of the old cuticle at the end of the molt), CCAP may control the level of activity of the anterograde cardiac pacemaker in the adult fly. Expression of double stranded CCAP RNA in the CCAP neurons (targeted CCAP RNAi) causes a significant reduction in CCAP expression. However, this reduction is not sufficient to compromise CCAP's function in ecdysis behavior and heartbeat regulation (Dulcis, 2005).

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).

Effects of Ccap neuron cellular knockout

To produce animals lacking Ccap neurons, expression of the cell death gene reaper (rpr) was driven in Ccap neurons, using the Ccap-GAL4 transgenic strain. A similar approach has been successfully used to study the function of other Drosophila neuropeptides and hormones. To investigate the consequences of loss of Ccap neurons on larval ecdysis, Ccap neurons had to be absent, at the latest, prior to the last larval ecdysis (from 2nd to 3rd larval instar). The targeted expression of rpr using the Ccap-GAL4 driver was shown to produce late 2nd instar larvae that are probably entirely devoid of Ccap function. In certain experiments that examined post-larval ecdyses, animals were transferred to 20°C after collection as first instar larvae and raised at this temperature until pupation or eclosion. At this lower temperature the vast majority of the CNSs was also mostly devoid of Ccap neurons by the end of the 3rd instar (at wandering). Thus, of 22 CNSs examined at this time, 15 showed no Ccap- or ß-gal-immunoreactive neurons or processes, while four, two and one CNSs had one, two and four weakly stained neurons, respectively, and none of these CNSs had visible immunoreactive processes. When the CNS of animals raised using the eclosion rhythm paradigm (25°C to 18°C) was processed for Ccap- and ß-gal-IR immediately after adult eclosion, 25 of 28 CNSs showed no immunoreactivity, while two and one CNSs had one and two weakly staining neurons, respectively, lacking visible processes (Park, 2003).

The role of Ccap in larval ecdysis

In the moth Manduca sexta, addition of CCAP to an isolated larval abdominal CNS turns on the ecdysis motor program (Gammie, 1997b; Zitnan, 2000). This, and other evidence (reviewed by Ewer, 2002), strongly implicates the CCAP neuropeptide in the control of ecdysis behavior in this moth. In the CNS of Drosophila larvae, Ccap-IR decreases shortly before the onset of larval ecdysis, suggesting that Ccap is similarly important for the control of ecdysis in this insect (Park, 2003).

In Drosophila, there are two larval intermolts, the intermolt between first and send larval instars, and the intermolt between second and third instars. To investigate directly the role of Ccap in larval ecdysis, animals lacking the Ccap neuronal population were characterized. Surprisingly, it was found that genetic ablation of the Ccap neurons is not lethal during the larval stages. Indeed, the survival rate of Ccap-KO from 1st instar to the end of the 3rd (last) instar is indistinguishable from that of the control population (97% vs. 95%, respectively). This indicates that Ccap is not essential for viability during (at least) the latter part of the 2nd larval intermolt period and the entire 3rd larval instar. Most significantly, animals lacking Ccap neurons were able to shed their old cuticle at the end of molt to the 3rd instar. Independent studies show that animals homozygous for small chromosomal deletions including Ccap (and 14 other genes) survive until the 3rd instar. Thus, survival of Ccap-KO larvae until this stage is not due to persisting (but immunohistochemically undetectable) Ccap peptide (Park, 2003).

To determine whether ecdysis behavior is normal in KO animals, the sequence and timing of ecdysis behavior from the 2nd to the 3rd instar was examined. The earliest obvious marker for the impending ecdysis is the appearance of pigmentation in the mouth plates of the future 3rd instar (double mouth plates stage; DMP), which occurs about 30 minutes before ecdysis. Approximately 16 minutes after the DMP stage, air enters the new trachea; this is followed shortly by the onset of the preparatory behavior called pre-ecdysis ( Park, 2002a). Approximately 15 minutes after air filling, pre-ecdysis stops and the animal executes a characteristic 'biting' behavior during which it appears to be attempting to tear the anterior region of the old cuticle. This period is then followed by the onset of ecdysis proper, which is characterized by vigorous peristaltic waves sweeping along the animal in a posterior-to-anterior direction. Typically after three to four waves, the anterior cuticle breaks, freeing the 3rd instar of its 2nd instar cuticle. After a period of a few minutes the animal resumes feeding and locomotory behavior (Park, 2003).

Although the Ccap-KO larvae are clearly able to initiate ecdysis behavior and use this behavior to free themselves from the 2nd instar cuticle, there are subtle but significant differences between the behavior of Ccap-KO and control animals. The duration of events up to the onset of pre-ecdysis is indistinguishable for these two groups of larvae. The first notable difference between Ccap-KOs and control larvae is a modest but significant lengthening of pre-ecdysis behavior, although pre-ecdysis behavior itself appears normal (Park, 2003).

Additionally, the subsequent events that lead to cuticle shedding takes approx. three times longer in Ccap-KO larvae than in controls. Both the biting period, which occurs between the end of pre-ecdysis and ecdysis onset, and the duration of ecdysis itself, are significantly extended in Ccap-KO animals. Interestingly, Ccap-KO larvae exhibit anterior to posterior peristaltic waves interspersed with the typically occurring posterior to anterior waves, a behavior never observed in control animals. Because the waves moving in the anterior to posterior direction do not aid in breaking the old cuticle, the time to successful shedding of the old cuticle is lengthened (Park, 2003).

These results reveal that the ablation of Ccap neurons is associated with defects that are strictly confined to the execution of ecdysis itself. Thus, while the entire duration of the period between DMP and ecdysis is increased by only 14%, from the normal 31.9±1.0 minutes to 36.3±0.9 minutes, the timing and organization of ecdysis behavior itself is quite severely disrupted in the KO animals (Park, 2003).

The role of Ccap in pupal ecdysis

In higher Diptera such as Drosophila , pupal ecdysis (pupation) corresponds to the behavior referred to as 'head eversion'. During head eversion, the brain, which in the larva is located behind the mouthparts, is pushed anteriorly to a position in front of the thorax and the mouthparts. At the same time, the appendages, which were formed from the eversion of the imaginal discs at pupariation (the onset of the larval-pupal transition), are extended to attain their final size and shape (Park, 2003).

In contrast to the situation observed in the larva, most Ccap-KO animals die during the pupal stage. Furthermore, the appearance of KO animals at the end of pupation and of metamorphosis suggests that the primary cause of their death is a specific failure of pupal ecdysis. Indeed, in KO pupae and pharate adults, the head is located much more to the anterior than normal or is only partially everted; the larval tracheae have not been completely shed; and the appendages have not been properly extended, resulting in a pharate adult that has abnormally short wings and legs (Park, 2003).

In order to determine the bases for these defects, the timecourse of pupal ecdysis was examined in KO animals. In Drosophila , pupation occurs approx. 12 hours after pupariation. Pupal ecdysis is preceded by a preparatory behavior (termed here pre-ecdysis by analogy to the corresponding larval behavior), during which the posterior part of the animal rhythmically retracts from the puparium. About 9-10 minutes after the onset of pre-ecdysis, a short succession of peristaltic waves sweeps from the posterior to the anterior of the animal and causes the eversion of the head, the shedding of the larval tracheae, and a rapid extension of the appendages. Head eversion is followed by a long post-ecdysis period of several hours during which regular contractions, primarily of the abdomen, occur; this presumably aids in giving the insect its final form (Park, 2003).

Ccap-KO animals initiate normal pre-ecdysis behavior (for instance the frequency of abdominal 'sweeps' is the same as in controls). However, this behavior lasts significantly longer than in controls and is not followed by head eversion. Instead, abdominal pre-ecdysis movements eventually cease during a final retraction and are followed by a period that resembles the postecdysis period seen in the control (but which is significantly shorter in Ccap-KO animals (Park, 2003).

Although the KO pre-pupae all lack Ccap neurons, the morphological phenotype seen at the end of metamorphosis is somewhat variable, with, for instance, a variable amount of the adult head visible at the end of adult development. However, all flies show shortened appendages, and all animals whose pupation behavior was observed in detail show no pupal ecdysis. The basis for this variable phenotype is currently unknown (Park, 2003).

If head eversion is stimulated by Ccap, the neuropeptide should be released at this time. Indeed, in wild type animals, a substantial decrease in Ccap-IR is detected following pupation in descending Ccap immunopositive axons. The slight increase in the number of Ccap-immunoreactive varicosities that is apparent at the start of pre-ecdysis is a reflection of a subtle fragmentation in the pattern of Ccap-IR that is seen at this time, and may be the first sign that Ccap has started to be released. The ETH peptides are known to be essential for larval pre-ecdysis in Drosophila (Park, 2002a), and the drop in ETH-IR that is observed at the onset of pupal pre-ecdysis suggests that these peptides may also control this behavior at pupation (Park, 2003).

The role of Ccap in adult ecdysis (eclosion)

The KO animals that formed relatively normal heads at the end of metamorphosis are usually able to exit from the pupal case. A careful examination of eclosion has shown that the developmental and behavioral events that take place at this time occurs in the correct sequence in Ccap-KO animals, although some quantitative differences in the duration or number of events are observed. Thus, while tracheal filling occurs before the start of the eclosion behaviors, in Ccap-KO animals it takes longer than in controls. However, the ptilinum, which is used to rupture the anterior of the pupal case, is deployed normally at the expected time. Finally, and most significantly, the bouts of rapid anteriorly directed peristalses of ecdysis proper occurred in the KO animals. Interestingly, however, these bouts are relatively ineffective at propelling the animal forward. This is not due to a difference in the characteristics of the bouts themselves, which careful cinematographic analyses shows are very similar to those of control animals. Instead, this failure occurred because the abdomen of KO animals is not distended at this time, severely reducing the traction exerted by the body on the inner walls of the pupal case: this is needed in order for the abdominal peristalses to cause the rapid net forward movement that is seen in control animals. Although most KO animals (nine out of 10 examined) eventually succeeded in eclosing, extrication took much longer than normal. Thus, unlike larval and pupal ecdysis, the actual ecdysis motor program of the adult appears to be relatively normal in KO animals (however, the frequency of peristalses is lower than in controls, even when the two groups are compared during the first minute after the onset of eclosion, which corresponds approximately to the duration of eclosion in controls. Therefore, the most dramatic deficiency of Ccap-KO animals at eclosion appears to be due to the absence of a function required to expand the body rather than to a failure in the adult ecdysis motor program itself (Park, 2003).

The adult phenotype

The phenotypes of adult Ccap-KO flies suggest that Ccap neurons play some role in post-eclosion events. KO adults do not inflate their wings, and their cuticle appears to remain soft and untanned, as evidenced by the dimpling that is observed on the dorsal thorax at sites of thoracic muscle insertion. The defect in wing expansion may be due, in part, to the failure in wing extension at the time of pupation (Park, 2003). The tanning defect of the KO flies may occur because a subset of the Ccap neurons expresses the gene encoding the tanning hormone, bursicon (E. Dewey and H. W. Honegger, personal communication to Park, 2003).

In another experiment, the Ccap-GAL4 driver was used to overexpress a temperature-sensitive form of shibire (shi; the fly dynamin homolog) in the CCAP cell population (using a UAS-shi^ts transgene). When reared at 29°C, progeny carrying both the Ccap-GAL4 and UAS-shi^ts transgenes exhibited defects in wing expansion ~80%-100% of the populations), whereas control progeny (with only one transgene) have normal wings. At 25°C, both types of progeny have normal wings, indicative of a temperature-sensitive effect (Park, 2003).

Eclosion rhythms in the absence of Ccap neurons

To examine circadian rhythms of eclosion, Ccap-KO and control animals were reared under conditions that produced the maximal number of pharate adults, and then adult emergence was scored at two-hour intervals over the course of several days, both under a light:dark cycle (LD 12:12) and in constant darkness (DD). In three separate experiments using one Ccap-GAL4 line and in two separate experiments using a second independent transgenic line, a clear rhythmicity was observed under both LD and DD conditions, with most of the animals eclosing in the dawn-early morning (or subjective dawn-early morning) interval. Nevertheless, there were differences between the eclosion profiles of KO and control populations. Most notably, the temporal gate of eclosion was lengthened in KO animals, with significant emergence occurring in the late night/predawn period. Coupled with this wider eclosion 'gate', a significant diminution was observed in the amplitude of the eclosion burst that occurs immediately following lights-on, which in control populations constitutes approximately 40% of the flies that emerge on any given day. No consistent difference in the peak time of eclosion was observed between KO and control populations in LD or DD conditions (Park, 2003).

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).

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).

Effects of Mutation or Deletion

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).

Nutrients and pheromones promote insulin release to inhibit courtship drive

Food and reproduction are the fundamental needs for all animals. However, the neural mechanisms that orchestrate nutrient intake and sexual behaviors are not well understood. This study found that sugar feeding immediately suppresses sexual drive of male Drosophila, a regulation mediated by insulin that acts on insulin receptors on the courtship-promoting P1 neurons. The same pathway was co-opted by anaphrodisiac pheromones to suppress sexual hyperactivity to suboptimal mates. Activated by repulsive pheromones, male-specific PPK23 neurons on the leg tarsus release crustacean cardioactive peptide (CCAP) that acts on CCAP receptor on the insulin-producing cells in the brain to trigger insulin release, which then inhibits P1 neurons. These results reveal how male flies avoid promiscuity by balancing the weight between aphrodisiac and anaphrodisiac inputs from multiple peripheral sensory pathways and nutritional states. Such a regulation enables male animals to make an appropriate mating decision under fluctuating feeding conditions (Zhang, 2022).

This work identified a neuropeptidergic neural circuit underlying mating decision, and a direct link is revealed between the 'metabolic center' and the 'sex center' in the Drosophila brain. First, sugar feeding largely suppressed male sexual drive toward a virgin female, and this metabolic state-dependent neural control relied on ILP2 and ILP5. A suppression on P1 neurons was induced in vitro with the activation of IPCs, which was triggered by loading sugar. This conclusion was further validated by the fact that the decrease of P1 neuron activity was weaker when InR was knock down specifically on P1 neurons. It was also demonstrated that leg tarsal PPK23 M cells release CCAP once contacting aversive pheromone and this inhibitory signal activates IPCs in the brain via CCAP-R to release ILP2/5. IPC-derived ILP2/5 furthermore suppresses the activity of courtship-promoting P1 neurons to ultimately shut down male's sexual drive toward inappropriate mating targets. Together with inhibition on P1 by the tandem connections of PPK23 M cells and mAL neurons, male flies maintain high selectivity against inappropriate mates (Zhang, 2022).

As the central hub in control of male courtship, P1 neurons in the brain integrate both the external and internal information. The former includes sensory inputs from a potential mating target. While visual and olfactory cues are reported to trigger a male's propensity to court, contact pheromones can be either attractive or repulsive and gate the perception for other sensory pathways. The internal state, on the other hand, represents the male's readiness to mate, e.g., his mating history and nutritional state. P1 neurons constantly monitor the internal state of a male to evaluate the readiness to court or mate. For example, dopaminergic neurons control the mating drive of a male, and neuropeptide Drosulfakinin (DSK) and NPF impose an inhibitory tone on P1 neurons by encoding either mating experiences or nutritional state. In addition, sleep regulates mating via the functional interaction between circadian neurons and P1. Insulin signaling reportedly plays a well-established role in homeostatic regulation. This study revealed an unprecedented role of insulin in modulating male sexual behavior. On the basis of the fact that insulin level changes under numerous physiological conditions, such as feeding, temperature change, and sleep, the current findings raised the question whether, under such conditions, insulin regulates sexual activity via InR on P1. The present data support the existence of feeding/mating interaction via the insulin signaling (Zhang, 2022).

Further investigation is needed to reveal the biological significance of courtship inhibition immediately after a sugar meal. In the studies of human and mouse, sugar intake reduces the level of testosterone, an effect likely mediated with insulin. This adversity of sugar intake on libido may be important for the animals' fitness. There is an immediate boost of the blood sugar level after a sugar meal. Insulin is then secreted to help move the sugar from the blood into the cells. During this process, the sexual activity may be momentarily inhibited to foster sugar uptake. It still needs further investigation how decreased sexual behaviors contribute to energy storage. Another interesting question is whether other nutrition-related hormones such as Adipokinetic hormone (Akh) and Unpaired 2 (Upd2) also regulate sexual activity in flies. It is already reported that DSK and NPF are both required to tune a male's sexual drive. Recently, it was reported that protein intake caused postprandial sleepiness that may be critical for protein metabolism, suggesting that animals' nutritional homeostasis is critical in maintaining the balance of their feeding, reproduction, and other behaviors (Zhang, 2022).

Another intriguing question that warrants further investigation is whether insulin released upon contacting aversive pheromones would cause certain metabolic consequences to the males. It was reported that exposing male flies to female pheromone but preventing them from mating reduced males' life span. As deterring courtship is relatively fast, it would be interesting to look at the long-term effects after a male is exposed to aversive pheromones (Zhang, 2022).

Insulin was implicated to regulate female sexual receptivity. The female flies with mutations in their insulin-like protein genes exhibit a higher sexual activity, a similar defect as seen in male flies in current study. However, the neural circuit controlling mating in male and female shows profound sexual dimorphism. It thus raises an interesting question: How does insulin regulate the sexual drive in females? A virgin female's receptivity is controlled by double-sex-positive neurons in the brain. Among them, clusters of neurons of pCd and pC1 play a determinant role in female's sex behaviors. It is still an open question whether these neurons express InR and, if so, under what circumstance are they inhibited by insulin release. Insulin is essential for vitellogenesis in female flies, suggesting that insulin signaling may play differential roles at different reproductive stages (Zhang, 2022).

REFERENCES

Search PubMed for articles about Drosophila Crustacean cardioactive peptide

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date revised: 1 March 2024

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