InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

Feeding is a fundamental activity of all animals that can be regulated by internal energy status or external sensory signals. A zinc finger transcription factor, klumpfuss, is required for food intake in Drosophila larvae. Microarray analysis indicates that expression of the neuropeptide gene hugin (hug) in the brain is altered in klu mutants and that hug itself is regulated by food signals. Neuroanatomical analysis demonstrates that hug-expressing neurons project axons to the pharyngeal muscles, to the central neuroendocrine organ, and to the higher brain centers, whereas hug dendrites are innervated by external gustatory receptor-expressing neurons, as well as by internal pharyngeal chemosensory organs. The use of tetanus toxin to block synaptic transmission of hug neurons results in alteration of food intake initiation, which is dependent on previous nutrient condition. These results provide evidence that hug neurons function within a neural circuit that modulates taste-mediated feeding behavior (Melcher, 2005).

In a screen for Drosophila mutant larvae defective in feeding, the P-element line P(9036) was identified. These animals fail to pump food from the pharynx into the esophagus; this is not due to a morphological block in the esophagus. The failure to feed is also not due to a general illness of the animal or global locomotory defects, because they can move around with the same vigor as wild-type or heterozygote siblings. P(9036) larvae also display wandering-like behavior, in which they move away from the food. During this wandering-like phase, P(9036) larvae move about with food lodged in their pharynx, further supporting the view that the feeding defect is not due to a general body movement defect. Wandering behavior is observed in wild-type larvae when they stop feeding and move away from food shortly before pupariation. These feeding behavior defects have also been observed for pumpless (ppl) mutants. ppl encodes an amino acid catabolizing enzyme that is expressed exclusively in the fat body, an organ analogous to the vertebrate liver. Thus, P(9036) and ppl mutants, as immature first instar larvae, display feeding behaviors characteristic of sated, full-grown, third instar larvae. The gene corresponding to P(9036) was characterized and found to be klu, a zinc finger protein-encoding gene that is expressed specifically in the developing nervous system. P(9036) fails to complement the lethality of all klu alleles tested, and trans-heterozygotes also show the characteristic feeding defect (Melcher, 2005).

To study the central control process that could underlie the feeding defect of klu mutants, microarray analysis of klu mutants was performed with a focus on neuropeptide genes. It was reasoned that their expression patterns in the brain would be specific enough for analysis at single-cell resolution. Furthermore, neuropeptides have been shown to influence food intake in different organisms, including mammals. RNA from klu mutant larvae and wild-type larvae were isolated and hybridized to three Affymetrix chips each and compared. In situ hybridizations were performed on wild-type larval brains with the six highest upregulated genes. Efforts were focused on hug because it had the most specific expression pattern in the larval brain. While all others showed staining in different parts of the brain or in the ventral nerve cord (VNC), hug showed staining in only a cluster of about 20 cells in the subesophageal ganglion (SOG) of the larval brain, with no staining anywhere else. hug expression in embryos is also highly restricted in the brain. hug encodes a prepropeptide capable of generating at least two neuropeptides, Drm-PK2 and hug-γ. The former encodes a myostimulatory peptide while the latter shows homology to ecdysis-triggering hormone-1, ETH-1. Both can activate a G-protein-coupled receptor belonging to the vertebrate neuromedin U group. A hug homolog is also found in Anopheles gambiae (Melcher, 2005).

To confirm the microarray data, semi-quantitative in situ hybridization was performed in wild-type and klu mutant larval brains. hug is upregulated in klu mutants. Whether hug expression is also regulated in ppl larvae, which display a similar feeding defect as klu, was examined. There is also an upregulation of hug in ppl mutants. Whether hug expression is regulated by different nutrient signals was examined. Wild-type larvae were placed in starvation and sugar-rich conditions (that is, both being amino acid-deficient diets) and hug expression was monitored. hug was downregulated in both conditions, indicating a response to nutrient signals distinct from simple lack of energy. Since hug is upregulated in klu and in ppl mutants, both of which do not feed and wander about, a higher hug level correlates with decrease of food intake and food-seeking behavior. Under starvation and sugar conditions, a lower hug level correlates with increased food-seeking behavior, since larvae become hyperactive and disperse when food is removed (Melcher, 2005).

The identification of candidate chemosensory receptors in mammals and invertebrates has provided major insights into the molecular mechanisms underlying sensory information processing. In the Drosophila olfactory system, projections of OR-expressing sensory organs terminate at specific glomerular structures in the antennal lobe. The olfactory projection neurons then act in a second relay to convey the information to the mushroom bodies in the higher brain region. The gustatory organs, expressing specific GRs, project to a different brain region, the SOG, which has been implicated in gustatory signal transduction and feeding response in different insects. The current results indicate that the neurons that express the hug neuropeptide gene are likely candidates for acting as interneurons that transduce gustatory information. These comprise an assembly of about 20 neurons in the SOG. The close proximity of their dendrites with the axon terminals of gustatory sensory organs of the external head, and chemosensory organs of the internal pharynx, suggests a synaptic contact, but this requires functional verification. Whether the SOG is also organized into glomerular structure, like the antennal lobe, is not known. Such an organization has been suggested in adult Drosophila, although data on larvae have been lacking. The results on the dendritic pattern of hug neurons also suggest a glomerular structure of the larval SOG, but this remains an open issue (Melcher, 2005).

The hug neurons, in turn, send axons to at least three distinct targets: the ring gland, the pharyngeal muscles, and the protocerebrum. The projections to the ring gland and the pharyngeal muscles suggest that hug neurons coordinate sensory information with growth, metabolism, and food intake; the axon tracts to the protocerebrum suggest a role of hug neurons in transducing sensory signals for processing in the higher brain centers. These axon tracts are distinct from those of the olfactory projection neurons, projecting to a more dorsomedial region of the mushroom body, and adjacent to the median neurosecretory cells of the pars intercerebralis. Thus, hug neurons are ideally connected to undertake the role of integrating gustatory sensory signals with higher brain functions and feeding behavior (Melcher, 2005).

The chemosensory systems of all animals play critical roles in modulating feeding behavioral response. Feeding behavior can have diverse aspects, including locating a food source, evaluating food for nutritional appropriateness, choosing between different food sources, and deciding to initiate or terminate feeding. Blocking synaptic transmission by tetanus toxin in the hug neurons alters a specific aspect of the feeding behavioral response. When transferred to a certain new food source, the control flies wait for a period before initiating feeding, whereas experimental flies start feeding almost immediately. In both cases, the size of the crop after a longer feeding period does not change, meaning that no difference is seen in the termination phase of feeding. It is interesting to note that GR66C1 (also named GR66a) neurons, which project to hug dendrites, have recently been shown to mediate aversive taste response (Wang, 2004). This is consistent with the behavior of flies in which hug signaling is decreased, since they lose their 'aversive' response, as manifested in the elimination of a wait period before feeding. This behavior is dependent on internal nutrient status, as well as food quality, since if animals are starved or given food with an aversive tastant beforehand (such as yeast with quinine), control flies also start feeding immediately on the new yeast source (Melcher, 2005).

Insects have evolved a wide variety of feeding behaviors based on food identity, quality, and availability. Some of these are innate, whereas others are acquired through experience. For example, food preference in the tobacco hornworm is dependent on what they initially encounter after hatching. They are capable of growing on a wide variety of sources, but once they have fed on a particular food type, they will maintain this food preference. In this context, a possible scenario is that Drosophila associate feeding with a particular food source with which they become familiar. When they encounter a different food source, they must first re-evaluate it, perhaps for nutrient content, or adapt to it, before initiating feeding. Therefore, hug neurons appear to regulate the decision to initiate feeding based on previous food experience (Melcher, 2005).

In animals with a developed endocrine system, there is an intricate interdependence among feeding, growth, and neuroendocrine activity. Drosophila larvae are characterized by continuous feeding and a huge increase in organismal growth; in the adult, although no growth at organismal level takes place, a large cellular growth is required in the female for egg production. Both are highly dependent on feeding and the quality of food, such as protein content, and are under neuroendocrine control. klu and ppl represent two genes that are required for food intake and growth in Drosophila. Mutations in both genes result in reduced food intake and growth. In addition, as young larvae, mutants display a wandering-like behavior, which is reminiscent of full-grown wild-type larvae, which stop feeding and move away from the food source just prior to pupariation, a process dependent on the neuroendocrine system. Mutations in either of the genes lead to an upregulation of hug neuropeptide gene expression in the brain, whereas hug expression is downregulated in the absence of food signals (Melcher, 2005).

What could be the function of the hug neuropeptides? hug encodes at least two distinct neuropeptides. One (hug-γ) has homology to an ecdysone triggering hormone, while the second (Drm-PK-2) is a pyrokinin with myostimulatory activity. hug-γ could be involved in controlling growth and metabolism. This view is supported by projection of hug neurons to the ring gland, the master neuroendocrine organ. In addition, overexpression of hug has been shown to cause molting defects (Meng, 2002). Drm-PK-2, in contrast, may play a role in modifying the mechanical aspect of food intake, which is supported by the projection of hug neurons to the pharyngeal muscles. One interesting possibility is that the different neuropeptides are translated or trafficked to different targets in subset of hug neurons. In this case, a common gene expression pattern can be utilized to send out different signals to the different targets, such as to the higher brain center, feeding apparatus, and neuroendocrine organ. This would be a mechanism for coordinating different growth-dependent processes with a common input signal, for example, from a particular food signal. In this context, one way to explain the upregulation of the hug gene in klu and ppl mutants would be that the level of hug gene differentially correlates with the degree of food-seeking response. High levels, as in the mutants that do not feed, would reflect lower feeding and food-seeking response, whereas low levels, as in the absence of food sensory input, would reflect increased food-seeking response. This would also be consistent with hug overexpression studies and with the correlation seen between decreasing hug neuronal activity and increased feeding. Further functional studies, including imaging analysis, should increase understanding of how the hug neural circuit coordinates sensory perception, feeding behavior, and growth (Melcher, 2005).

Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophila brain

Bitter is a taste modality associated with toxic substances evoking aversive behaviour in most animals, and the valence of different taste modalities is conserved between mammals and Drosophila. Despite knowledge gathered in the past on the peripheral perception of taste, little is known about the identity of taste interneurons in the brain. This study shows that hugin neuropeptide-containing neurons in the Drosophila larval subesophageal zone are necessary for avoidance behaviour to caffeine, and when activated, result in cessation of feeding and mediates a bitter taste signal within the brain. Hugin neuropeptide-containing neurons project to the neurosecretory region of the protocerebrum and functional imaging demonstrates that these neurons are activated by bitter stimuli and by activation of bitter sensory receptor neurons. The study proposes that hugin neurons projecting to the protocerebrum act as gustatory interneurons relaying bitter taste information to higher brain centres in Drosophila larvae (Hückesfeld, 2016).

The bitter taste rejection response is important for all animals that encounter toxic or harmful food in their environment. This study showed that the hugin neurons in the Drosophila larval brain function as a relay between bitter sensory neurons and higher brain centres. Strikingly, activation of the hugin neurons, located in the subesophageal zone, made the animals significantly more insensitive to substrates with negative valence like bitter (caffeine) and salt (high NaCl), as well as positive valence like sweet (fructose). In other words, when the hugin neurons are active these animals 'think' they are tasting bitter and therefore become insensitive to other gustatory cues. This is in line with observations made in mice, in which optogenetically activating bitter cortex neurons caused animals to avoid an empty chamber illuminated with blue light. In this situation, although mice do not actually taste something bitter, they avoid the empty chamber since the bitter perception has been optogenetically induced in the central nervous system (CNS) and the mice 'think' they are tasting a bitter substance (Hückesfeld, 2016).

In previous work, activation of all hugin neurons led to behavioural and physiological phenotypes such as decreased feeding, decrease in neural activity of the antennal nerve (AN), and induction of a wandering-like behaviour (Schoofs, 2014). The neurons responsible specifically to those that project have now been pinpointed to the protocerebrum. These neurons not only respond to bitter stimuli, but also show a concentration dependent increase in calcium activity in response to caffeine. Dose dependent coding of bitter taste stimuli was previously shown to occur in peripheral bitter sensory neurons, where bitter sensilla exhibit dose dependent responses to various bitter compounds. Larvae in which the huginPC neurons have been ablated still showed some avoidance to caffeine. Whether this implies the existence of other interneurons being involved in caffeine taste processing remains to be determined. Interestingly, the huginPC neurons are inhibited when larvae taste other modalities like salt (NaCl), sugar (fructose) or protein (yeast). This may indicate that taste pathways in the brain are segregated, but influence each other, as previously suggested (Hückesfeld, 2016).

Bitter compounds may be able to inhibit the sweet-sensing response to ensure that bitter taste cannot be masked by sweet tasting food. This provides an efficient strategy for the detection of potentially harmful or toxic substances in food. For appetitive tastes like fructose and yeast, bitter interneurons neurons like the huginPC neurons in the CNS may become inhibited to ensure appropriate behaviour to pleasant food. Salt is a bivalent taste modality, that is, low doses of salt drive appetitive behaviour, whereas high doses of salt are aversive to larval and adult Drosophila. Inhibition of huginPC neurons when larvae are tasting salt might be due to a different processing circuit for different concentrations of salt and the decision to either take up low doses or reject high doses (Hückesfeld, 2016).

Taken together, it is proposed that hugin neuropeptide neurons projecting to the protocerebrum represent a hub between bitter gustatory receptor neurons and higher brain centres that integrate bitter sensory information in the brain, and through its activity, influences the decision of the animal to avoid a bitter food source. The identification of second order gustatory neurons for bitter taste will not only provide valuable insights into bitter taste pathways in Drosophila, but may also help in assigning a potentially novel role of its mammalian homologue, Neuromedin U, in taste processing (Hückesfeld, 2016).

REGULATION

Blocking synaptic transmission of hug neurons alters food intake behavior

Based on the connectivity map of hug neurons and the alteration in hug expression under different nutrient and feeding conditions, a series of experiments was initiated to explore the role of hug in regulating feeding. Since hug mutants have not yet been identified, the effects of overexpressing hug in the larvae was tested. hugS3 was used to drive hug expression but no phenotype was observed. This is most likely because using an endogenous promoter does not result in high enough overexpression of hug in cells that already express physiological levels of hug. Therefore a strong ubiquitous promoter (tubulin-gal4) was used. There was a strong reduction in growth, with no larvae surviving to pupal stage; defects in food intake were observed, although not to the same strong degree as with klu mutants. This is consistent with the view that high hug levels correlate with decreased food intake (Melcher, 2005).

In order to gain further information on the function of hug neurons, synaptic transmission in these cells was blocked using tetanus toxin light chain (TeTxLC). The experiments were carried in the larvae but no difference was seen. However, it was reasoned that any potential increase in feeding response may not be readily detectable in the larvae because they feed continuously, already at a maximal rate. Therefore, whether blocking synaptic transmission of hug neurons could suppress the feeding defect of klu mutants was tested. A significant rescue was observed of klu mutant feeding phenotype (Melcher, 2005).

Behavioral analysis on adults was carried out, since they are discontinuous feeders and thus may display an increased feeding behavior. Furthermore, one can visualize the quantity of food eaten by the size of the crop. Experimental and control flies were placed in food vials containing standard fly food for several days. They were then transferred to yeast paste containing red dye. A striking result was observed. After 5 min, the experimental flies had a completely filled crop, whereas the control lines had very little food in the crop. Even after 30 min, the control flies had very little food in their crops and only traces of red food were detectable in the midgut. By 180 min, both experimental and control flies showed the same degree of feeding. These results suggested that hug neurons are involved in regulating the initiation phase of feeding: control flies wait for a certain period before initiating feeding on the new food source, whereas decreasing hug neuronal signaling results in flies initiating their feeding immediately. When flies were transferred from yeast to colored yeast, or normal food to colored normal food, no difference was seen between experimental and control flies, indicating that hug neurons are not simply affecting the rate of feeding per se; no a difference was observed when transferring from yeast to normal food, indicating that the hug neuron-dependent behavioral effect is also not due to a simple fact of changing food sources. When flies were transferred from normal food into yeast containing 1M quinine (quinine is an aversive tastant), experimental flies again filled their crops earlier than controls. However, when flies were kept on yeast containing 1M quinine, and then transferred to yeast without the quinine, both experimental and control flies filled their crops with the new yeast within 5 min; analogously, when flies were starved before placing them on red yeast, both control and experimental flies filled their crops at about the same rate. These results suggest that the quality of previous food condition plays a role in defining hug neuronal function. Taken together, these studies support the view that hug neurons act within a neural circuitry in the brain that modulates feeding behavior based on chemosensory and nutrient signals (Melcher, 2005).

Synaptic transmission parallels neuromodulation in a central food-intake circuit

NeuromedinU is a potent regulator of food intake and activity in mammals. In Drosophila, neurons producing the homologous neuropeptide hugin regulate feeding and locomotion in a similar manner. This study used EM-based reconstruction to generate the entire connectome of hugin-producing neurons in the Drosophila larval CNS (see EM reconstruction of hugin neurons and their synaptic sites). Hugin neurons were shown to use synaptic transmission in addition to peptidergic neuromodulation, and acetylcholine was identified as a key transmitter. Hugin neuropeptide and acetylcholine are both necessary for the regulatory effect on feeding. Subtypes of hugin neurons connect chemosensory to endocrine system by combinations of synaptic and peptide-receptor connections. Targets include endocrine neurons producing DH44, a CRH-like peptide, and insulin-like peptides. Homologs of these peptides are likewise downstream of neuromedinU, revealing striking parallels in flies and mammals. It is proposed that hugin neurons are part of an ancient physiological control system that has been conserved at functional and molecular level (Schlegel, 2016).

Almost all neurons in Drosophila are uniquely identifiable and stereotyped. This enabled identification and reconstruction of a set of 20 peptidergic neurons in an ssTEM volume spanning an entire larval CNS. These neurons produce the neuropeptide hugin and have previously been grouped into four classes based on their projection targets. Neurons of the same morphological class (a) were very similar with respect to the distribution of synaptic sites, (b) shared a large fraction of their pre- and postsynaptic partners and (c) in case of the interneuron classes (hugin-PC and hugin-VNC), neurons were reciprocally connected along their axons with other neurons of the same class. This raises the question why the CNS sustains multiple copies of morphologically very similar neurons. Comparable features have been described for a population of neurons which produce crustacean cardioactive peptide (CCAP) in Drosophila. The reciprocal connections as well as the overlap in synaptic partners suggest that the activity of neurons within each interneuron class is likely coordinately regulated and could help sustain persistent activity within the population. In the mammalian pyramidal network of the medial prefrontal cortex, reciprocal connectivity between neurons is thought to contribute to the network's robustness by synchronizing activity within subpopulations and to support persistent activity. Similar interconnectivity and shared synaptic inputs have also been demonstrated for peptidergic neurons producing gonadotropin-releasing hormone (GnRH) and oxytocin in the hypothalamus. Likewise, this is thought to synchronize neuronal activity and allow periodic bursting (Schlegel, 2016).

Previous studies showed that specific phenotypes and functions can be assigned to certain classes of hugin neurons: hugin-VNC neurons increase locomotion motor rhythms but do not affect food intake, whereas hugin-PC neurons decrease food intake and are necessary for processing of aversive gustatory cues. For hugin-RG or hugin-PH such specific functional effects have not yet been described. One conceivable scenario would be that each hugin class mediates specific aspects of an overarching 'hugin phenotype'. This would require that under physiological conditions all hugin classes are coordinately active. However, no evidence of such coordination was found on the level of synaptic connectivity. Instead, each hugin class forms an independent microcircuit with its own unique set of pre- and postsynaptic partners. It is thus predicted that each class of hugin-producing neurons has a distinct context and function in which it is relevant for the organism (Schlegel, 2016).

Data presented in this study provide the neural substrate for previous observation as well as open new avenues for future studies. One of the key features in hugin connectivity is the sensory input to hugin-PC, hugin-VNC and, to a lesser extent, hugin-RG. While hugin-PC neurons are known to play a role in gustatory processing, there is no detailed study of this aspect for hugin-VNC or hugin-RG neurons. Sensory inputs to hugin neurons are very heterogeneous, which suggests that they have an integrative/processing rather than a simple relay function (Schlegel, 2016).

Hugin neurons also have profound effects on specific motor systems: hugin-PC neurons decelerate motor patterns for pharyngeal pumping whereas hugin-VNC neurons accelerate locomotion motor patterns. For hugin-PC, this study has demonstrated that this effect is mediated by both synaptic and hugin peptide transmissions. For hugin-VNC, this effect is independent of the hugin neuropeptide, suggesting synaptic transmission to play a key role. Suprisingly, no direct synaptic connections to the relevant motor neurons were found. However, the kinetics of the effects of hugin neurons on motor systems have not yet been studied at a high enough temporal resolution (i.e., by intracellular recordings) to assume monosynaptic connections. It is thus well conceivable that connections to the respective motor systems are polysynaptic and occur further downstream. Alternatively, this may involve an additional non-synaptic (peptidergic) step. A strong candidate for this is the neuroendocrine system which this study has identified as the major downstream target of hugin-PC neurons. Among the endocrine targets of hugin, the insulin-producing cells (IPCs) have long been known to centrally regulate feeding behavior. It is not known if insulin-signaling directly affects motor patterns in Drosophila. Nevertheless, increased insulin signaling has strong inhibitory effects on food-related sensory processing and feeding behavior. Whether the neuroendocrine system is a mediator of the suppressive effects of hugin-PC neurons on food intake remains to be determined (Schlegel, 2016).

The first functional description of hugin in Drosophila was done in larval and adult, while more recent publications have focused entirely on the larva. One of the main reasons for this is the smaller behavioral repertoire of the larva: the lack of all but the most fundamental behaviors makes it well suited to address basic questions. Nevertheless, it stands to reason that elementary circuits should be conserved between larval and adult flies. To date, there is no systematic comparison of hugin across the life cycle of Drosophila. However, there is indication that hugin neurons retain their functionality from larva to the adult fly. First, morphology of hugin neurons remains virtually the same between larva and adults. Second, hugin neurons seem to serve similar purposes in both stages: they acts as a brake on feeding behavior - likely as response to aversive sensory cues. In larvae, artificial activation of this brake shuts down feeding. In adults, removal of this break by silencing of hugin neurons leads to a facilitation (earlier onset) of feeding. Such conservation of neuropeptidergic function between larval and adult Drosophila has been observed only in a few cases. Prominent examples are short and long neuropeptide F, both of which show strong similarities with mammalian NPY. The lack of additional examples is not necessarily due to actual divergence of peptide function but rather due to the lack of data across both larva and adult. Given the wealth of existing data on hugin in larvae, it would be of great interest to investigate whether and to what extent the known features (connectivity, function, etc.) of this system are maintained throughout Drosophila's life history (Schlegel, 2016).

A neural network is a highly dynamic structure and is subject to constant change, yet it is constrained by its connectivity and operates within the framework defined by the connections made between its neurons. On one hand, this connectivity is based on anatomical connections formed between members of the network, namely synapses and gap junctions. On the other hand, there are non-anatomical connections that do not require physical contact due to the signaling molecules, such as neuropeptides/-hormones, being able to travel considerable distances before binding their receptors. The integrated analysis in this study of the operational framework for a set of neurons genetically defined by the expression of a common neuropeptide, positions hugin-producing neurons as a novel component in the regulation of neuroendocrine activity and the integration of sensory inputs. Most hugin neurons receive chemosensory input in the subesophageal zone, the brainstem analog of Drosophila. Of these, one class is embedded into a network whose downstream targets are median neurosecretory cells (mNSCs) of the pars intercerebralis, a region homologous to the mammalian hypothalamus. Hugin neurons target mNSCs by two mechanisms. First, by classic synaptic transmission as the current data strongly suggest that acetylcholine (ACh) acts as transmitter at these synapses. Accordingly, subsets of mNSCs have been shown to express a muscarinic ACh receptor. Whether additional ACh receptors are expressed is unknown. Second, by non-anatomical, neuromodulatory transmission using a peptide-receptor connection, as demonstrated by the expression of hugin G-protein-coupled receptor PK2-R1 (CG8784) in mNSCs. Strikingly, while PK2-R1 is expressed in all mNSCs, the hugin neurons have many synaptic contacts onto insulin-producing cells but few to DMS and DH44 neurons. This mismatch in synaptic vs. peptide targets among the mNSCs suggests an intricate influence of hugin-producing neurons on this neuroendocrine center. In favor of a complex regulation is that those mNSCs that are synaptically connected to hugin neurons additionally express a pyrokinin-1 receptor (PK1-R, CG9918) which, like PK2-R1, is related to mammalian neuromedinU receptors. There is some evidence that PK1-R might also be activated by the hugin neuropeptide, which would add another regulatory layer (Schlegel, 2016).

The concept of multiple messenger molecules within a single neuron is well established and appears to be widespread among many organisms and neuron types. For example, cholinergic transmission plays an important role in mediating the effect of Neuromedin U (NMU) in mammals. This has been demonstrated in the context of anxiety but not yet for feeding behavior. There are, however, only few examples of simultaneous employment of neuromodulation and fast synaptic transmission in which specific targets of both messengers have been investigated at single-cell level. In many cases, targets and effects of classic and peptide co-transmitters seem to diverge. In contrast, AgRP neurons in the mammalian hypothalamus employ neuropeptide Y, the eponymous agouty-related protein (AgRP) and the small molecule transmitter GABA to target pro-opiomelanocortin (POMC) neurons in order to control energy homeostasis. Also, reminiscent of the current observations is the situation in the frog sympathetic ganglia, where preganglionic neurons use both ACh and a neuropeptide to target so-called C cells but only the neuropeptide additionally targets B cells. In both targets, the neuropeptide elicits late, slow excitatory postsynaptic potentials (EPSPs). It is conceivable that hugin-producing neurons act in a similar manner by exerting a slow, lasting neuromodulatory effect on all mNSCs and a fast, transient effect exclusively on synaptically connected mNSCs. Alternatively, the hugin neuropeptide could facilitate the postsynaptic effect of acetylcholine. Such is the case in Aplysia where a command-like neuron for feeding employs acetylcholine and two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Both peptides work cooperatively on a postsynaptically connected motor neuron to enhance EPSPs in response to cholinergic transmission (Schlegel, 2016).

In addition to the different timescales that neuropeptides and small molecule transmitters operate on, they can also be employed under different circumstances. It is commonly thought that low-frequency neuronal activity is sufficient to trigger fast transmission using small molecule transmitters, whereas slow transmission employing neuropeptides requires higher frequency activity. Hugin-producing neurons could employ peptidergic transmission only as a result of strong excitatory (e.g. sensory) input. There are, however, cases in which base activity of neurons is already sufficient for graded neuropeptide release: Aplysia ARC motor neurons employ ACh as well as neuropeptides and ACh is generally released at lower firing rates than the neuropeptide. This allows the motor neuron to function as purely cholinergic when firing slowly and as cholinergic/peptidergic when firing rapidly. However, peptide release already occurs at the lower end of the physiological activity of those neurons. It remains to be seen how synaptic and peptidergic transmission in hugin neurons relate to each other (Schlegel, 2016).

The present study is one of very few detailed descriptions of differential targets of co-transmission and the first of its kind in Drosophila. These finding should provide a basis for elucidating some of the intriguing modes of action of peptidergic neurons (Schlegel, 2016).

The mammalian homolog of hugin, neuromedinU (NMU), is found in the CNS as well as in the gastrointestinal tract. Its two receptors, NMUR1 and NMUR2, show differential expression. NMUR2 is abundant in the brain and the spinal cord, whereas NMUR1 is expressed in peripheral tissues, in particular in the gastrointestinal tract. Both receptors mediate different effects of NMU. The peripheral NMUR1 is expressed in pancreatic islet β cells in humans and allows NMU to potently suppress glucose-induced insulin secretion. The same study also showed that Limostatin (Lst) is a functional homolog of this peripheral NMU in Drosophila: Lst is expressed by glucose-sensing, gut-associated endocrine cells and suppresses the secretion of insulin-like peptides. The second, centrally expressed NMU receptor, NMUR2, is necessary for the effect of NMU on food intake and physical activity. In this context, NMU is well established as a factor in regulation of the hypothalamo-pituitary axis and has a range of effects in the hypothalamus, the most important being the release of corticotropin-releasing hormone (CRH). This study shows that a subset of hugin-producing neurons targets the pars intercerebralis, the Drosophila homolog of the hypothalamus, in a similar fashion: neuroendocrine target cells in the pars intercerebralis produce a range of peptides, including diuretic hormone 44 which belongs to the insect CRH-like peptide family. Given these similarities, it is proposed that hugin is homologous to central NMU just as Lst is a homologous to peripheral NMU. Demonstration that central NMU and hugin circuits share similar features beyond targeting neuroendocrine centers, e.g. the integration of chemosensory inputs, will require further studies on NMU regulation and connectivity (Schlegel, 2016).

Previous work on vertebrate and invertebrate neuroendocrine centers suggests that they evolved from a simple brain consisting of cells with dual sensory/neurosecretory properties, which later diversified into optimized single-function cells. There is evidence that despite the increase in neuronal specialization and complexity, connections between sensory and endocrine centers have been conserved throughout evolution. It is proposed that the connection between endocrine and chemosensory centers provided by hugin neurons represents such a conserved circuit that controls basic functions like feeding, locomotion, energy homeostasis and sex (Schlegel, 2016).

Indisputably, the NMU system in mammals is much more complex as NMU is found more widespread within the CNS and almost certainly involves a larger number of different neuron types. This complexity, however, only underlines the use of numerically smaller nervous systems such as Drosophila's to generate a foundation to build upon. Moreover, NMU/NMU-like systems may have similar functions not just in mammals and Drosophila but also other vertebrates such as fish and other invertebrates such as C. elegans. In summary, these findings should encourage research in other organisms, such as the involvement of NMU and NMU homologs in relaying chemosensory information onto endocrine systems, and more ambitiously, to elucidate their connectomes in order to allow comparative analyses of the underlying network architecture (Schlegel, 2016).

Drm-PK-2 has pyrokinin activity in a muscle bioassay

Both putative peptides encoded by the hugin gene, Drm-PK-2 and hugγ, were consecutively tested on the same hyperneural muscle preparations. Threshold values for myostimulatory effects of the peptides differed clearly. The hugγ peptide exhibited a very low potency; Drm-PK-2 had a threshold concentration of about 3×10⁻⁸ M, thus being slightly less active than the sequence-related pyrokinin Pea-PK-3. The response of Drosophila heart preparations to Drm-PK-2 was a moderate increase in frequency not exceeding 150% of the control, even if the concentration was as high as 10⁻⁶ M (threshold at about 10⁻⁹ M). In contrast, most preparations showed a clear increase in amplitude after pyrokinin application with a threshold as low as. These results show that Drm-PK-2 has pyrokinin activity (Meng, 2002).

Protein Interactions

Two groups of GPCRs have been characterized predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of -PRXamide (PRXa). 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 (CG14575, 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, 2002).

The database of the Drosophila Genome Project contains the sequences of two genes, CG8784 and CG8795, predicted to code for two structurally related G protein-coupled receptors. These genes have been cloned their coding parts were expressed in Chinese hamster ovary cells. Both receptors can be activated by low concentrations of the Drosophila neuropeptide pyrokinin-2. The precise role of Drosophila pyrokinin-2 (SVPFKPRLamide) in Drosophila is unknown, but in other insects, pyrokinins have diverse myotropic actions and are also initiating sex pheromone biosynthesis and embryonic diapause. Gene silencing, using the RNA-mediated interference technique, showed that CG8784 gene silencing caused lethality in embryos, whereas CG8795 gene silencing resulted in strongly reduced viability for both embryos and first instar larvae. In addition to the two Drosophila receptors, two probable pyrokinin receptors were identified in the genomic database from the malaria mosquito Anopheles gambiae. The two Drosophila pyrokinin receptors are the first invertebrate pyrokinin receptors to be identified (Rosenkilde, 2003).

DEVELOPMENTAL BIOLOGY

hug is expressed specifically in a small group of neurons in the SOG. To gain insight into the physiological processes that hug-expressing neurons (referred to as hug neurons) could be involved in, their connectivity pattern was determined. Therefore, a hug promoter-Gal4 line (hugS3) was constructed in order to express different versions of green fluorescent protein (GFP) marker genes for neuroanatomical studies. This approach revealed hug neuron projection to the ring gland. The ring gland, as the master neuroendocrine organ of Drosophila larvae, controls metabolism and growth. For example, median neurosecretory cells of the pars intercerebralis that express Drosophila insulin like peptides (dilps), also project to the ring gland, whereas Adipokinetic hormone (Akh), thought to be a glucagon homolog, is produced by the ring gland (Melcher, 2005).

In addition to the ring gland, hug neuron projection was observed to the protocerebrum, near the median neurosecretory cells and the mushroom bodies, which comprise the center for olfactory learning and memory. The axons projecting to the protocerebrum also cross at the midline just above the foramen. An intriguing glomerular-like structure was observed of what are most likely hug dendrites in the SOG, just dorsoanterior to the hug cell bodies. Glomerular organization in the SOG of adult Drosophila that relays gustatory information has been described. Such a glomerular organization has not been previously recognized for the larval SOG, but it would be analogous to the glomerular organization in the antennal lobes that relay olfactory information (Melcher, 2005).

Strikingly, there is also projection of hug neurons to the pharyngeal muscles, which pump food into the mouth atrium. These arise from axons that leave the brain and project anteriorly along each side of the dorsal pharyngeal muscles, and terminate near the anterior end of the pharynx. There has been no previous case of identified neurons in the larval SOG that project to motor outputs. At this point, it is not know whether the pharyngeal muscles are actually innervated by these axons. Taken together, these results demonstrate that hug neurons in the larvae project to key organs regulating feeding and growth-namely, the pharynx and the ring gland-as well as to higher brain centers (Melcher, 2005).

The projection of hug neurons to the mushroom body region, together with the fact that hug is expressed specifically in the SOG, which relays gustatory information, suggested that hug neurons could be involved in mediating chemosensory signals. Therefore, whether hug neurons receive direct input from the chemosensory organs was investigated. It has been demonstrated that sensory organs in the larval head that express ORs or GRs send their axons either to the antennal lobe or the SOG. Recently, an enhancer trap line MJ94 was used to label putative chemosensory organs of the internal pharynx (Gendre, 2004). As internal pharyngeal sensory organs are good candidates for transducing gustatory signals, it was asked if these sensory organs terminate at hug dendrites: they indeed terminate in the contact region of hug dendrites (Melcher, 2005).

To further test this, it was asked if chemosensory neurons that express specific GRs also project to hug dendrites. For example, it has been shown that GR66C1-positive neurons project to the SOG, whereas GR21D1-positive neurons project to the antennal lobe. To see if these sensory projections terminate at or near hug neurons, staining of GR66C1- and GR21D1-positive axon terminals was performed with hug in situ hybridization. GR66C1 receptor neurons indeed project to the vicinity of hug-expressing cells. To see if these axons may potentially make synaptic contacts with hug dendrites, a hug promoter-yellow fluorescent protein (YFP) line (in which YFP was placed directly under the hug promoter) was used in combination with GR promoters driving nSyb-GFP, thus allowing simultaneous visualization of GR axon terminals and hug dendrites. GR66C1-positive neurons project to the glomerular-like SOG region contacted by hug dendrites. GR21D1-positive neurons also project near the hug cells, but by contrast to GR66C1-positive neurons, do not contact hug dendrites. Rather, they terminate dorsoanterior to the hug dendrites, where the antennal lobes are located. Taken together, these results suggested that hug neurons may act as second-order interneurons that relay gustatory information (Melcher, 2005).

To further distinguish the relationship between hug neurons and the olfactory or gustatory systems, it was determined whether hug neurons share the same axon tracts to the mushroom bodies as the second-order neurons that relay olfactory sensory input. The dendrites of these olfactory projection neurons underlie the glomerular structure of the antennal lobes and vertically transduce olfactory information for processing to the mushroom bodies. These projections can be visualized by the enhancer trap line GH146. The axon projections of hug neurons are distinct from olfactory projection neurons: hug neurons project to a more dorsomedial region in the protocerebrum than olfactory projection neurons, and they use different axon tracts. These results essentially rule out hug neurons being olfactory projection neurons. Projection neurons transducing gustatory signals to higher brain centers have not yet been identified. In this context, hug neurons could act as gustatory projection neurons that connect gustatory sensory neurons via SOG with the protocerebrum (Melcher, 2005).

A difference was noticed in the projection specificity among the hug neurons. A series of enhancer trap lines have been isolated that label cells projecting their axons to the ring gland, one of which (Okt30) is co-expressed with hug. When the hug promoter-YFP line (to distinguish it from GFP reporter constructs) was used in combination with the Okt30 ring gland enhancer trap line, it was found that a distinct set of hug neurons project to only the ring gland and not to the protocerebrum, the pharynx, or the ventral cord (Melcher, 2005).

Another subpopulation of hug neurons is revealed by using the TH-Gal4 line. This drives reporter gene expression under the promoter of tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis. Specific hug neurons express TH-Gal4 reporter gene, indicating that a subset of hug neurons might be dopaminergic. When TH-Gal4-driven lacZ is used in combination with hug promoter-YFP, it was observed that TH-positive hug neurons project to only the pharynx and not to the protocerebrum, the ring gland, or the VNC. These results indicate that at least three distinct subpopulations of hug neurons exist: those projecting to only the ring gland, those projecting to only the pharyngeal muscles, and those projecting to the protocerebrum and/or the VNC. The distinct target specificity suggests differences in the function of the hug subpopulations. In the honeybee Apis, the subesophageal-calycal tract neurons are located in the SOG, send axons to the protocerebrum, and receive input from the sensory neurons of the proboscis; these neurons are thought to transduce gustatory information. Some of the hug neurons could act similarly to these honeybee neurons. Based on the connectivity map of the hug neurons, it was also of interest to find if the global targeting of these neurons was altered in klu mutant larvae. Therefore, the hug promoter-YFP construct was crossed into klu mutant background. Although it is not possible to rule out subtle local differences, the basic connectivity pattern is retained in the mutants (Melcher, 2005).

To see if hug neurons might also have a function in the adults, the connectivity pattern was determined in adult animals. There are some noticeable morphological differences in the feeding apparatus and neuroendocrine organs between adults and larvae. One is the presence of the crop in the adult but not in the larva. The crop is a food storage organ, and its absence in the larvae most likely reflects a difference in the feeding habits; whereas adults are intermittent feeders, the larvae feed continuously. Another is the relocation of the neuroendocrine organs. The corpora cardiaca/corpora allata (CC/CA) complex, which comprises part of the ring gland in the larvae, is located right above the proventriculus in the adults, at the junction between the gut and the crop. This is in contrast to the larvae, where it is located on top of the brain hemispheres. Monitoring hugS3 expression in adults, axon projections to the protocerebrum, the CC/CA complex, and the ventral cord were observed. A subpopulation of hug neurons may also be dopaminergic, as in the larvae. To further characterize the projections to the protocerebrum, the OK107 enhancer trap Gal4 line was used together with hug promoter-YFP. These stainings indicate that hug axons traverse along the median neurosecretory cells in the pars intercerebralis, and terminate near the mushroom bodies. The precise targets of hug neurons projecting to the protocerebrum remain to be determined. Taken together, despite the morphological differences, the connectivity pattern of hug neurons is remarkably similar between larvae and adults (Melcher, 2005).

Distinct classes of hugin-expressing neurons

The hugin gene of Drosophila encodes a neuropeptide with homology to mammalian neuromedin U. The hugin-expressing neurons are localized exclusively to the subesophageal ganglion of the central nervous system and modulate feeding behavior in response to nutrient signals. These neurons send neurites to the protocerebrum, the ventral nerve cord, the ring gland, and the pharynx and may interact with the gustatory sense organs. In this study, the morphology of the hugin neurons were investigated at a single-cell level by using clonal analysis. Single cells project to only one of the four major targets (see Melcher, 2007 for an illustration of the connectivity of hugin neurous). In addition, the neurites of the different hugin cells overlap in a specific brain region lateral to the foramen of the esophagus, which could be a new site of neuropeptide release for feeding regulation. This study reveals novel complexity in the morphology of individual hugin neurons, which has functional implication for how they coordinate feeding behavior and growth (Bader, 2007).

Each hugin neuron projects to only one of four major targets. Moreover, it this study reveals complexities in neuromorphology that have not been apparent before. Most notably, all hugin neurons arborize in the subesophageal ganglion (SOG) just lateral and ventral to the foramen, an opening in the Drosophila brain where the esophagus passes through. It is likely that this anatomical interface between CNS and gastrointestinal tract represents a local site for neuropeptide release. The single cell analysis additionally revealed close intermingling, precisely in the SOG, between some of the hugin neuronal projection termini and those of neurons mediating taste information from external and internal chemosensory organs. The gustatory receptor neuronal projections arborizing closest to hugin neurons are GR66a positive. Interestingly, this is a receptor that mediates aversive taste: if GR66a-expressing neurons are rendered nonfunctional by genetic manipulations, flies will feed on aversive substances, such as quinine and caffeine, and artificial activation of GR66a taste neurons elicits avoidance behavior to neutral substances (Bader, 2007, and references therein).

The hugin gene is expressed exclusively in 20 neurons in the SOG of the Drosophila larva, where they project to four distinct targets: the protocerebrum, the ventral nerve cord, the ring gland, and the pharynx. To determine the morphology of individual hugin neurons, the flp out technique was used to generate single cells marked with a fluorescent genetic marker. The details of each neurite target are described. Because it is not known at this point whether the different neurites are axons or dendrites, these terms have not been used in the descriptions (Bader, 2007).

Single clones of hugin cells were obtained that show neurites to the ipsilateral protocerebrum. These cells show arborization ventrolateral to the foramen of the esophagus in a region that is innervated by gustatory receptor neurons expressing GR66a. Connections have been observed between the left and the right protocerebrum, and clones were obtained in which neurites branch onto both hemispheres. Although no cells were observed that go only contralaterally, this is not proof of the nonexistence of such cells. Clones in which four cells on one side of the CNS all project to the ipsilateral protocerebrum. If one assumes the same for the other brain hemisphere, this implies that the protocerebrum is innervated by at least eight cells (Bader, 2007).

Single hugin cells also project down the ventral nerve cord. The morphology of these neurons is striking. In addition to a long process travelling down contralaterally along the lateral neuropile border of the ventral cord, there are four shorter fibers projecting up and down just left and right of the midline. The long neurites that extend down the lateral side of the ventral nerve cord branch out at the tip; the precise targets are not known. The two fibers that project in the anterior direction pass along each side of the foramen and end at the medial part of the protocerebrum. It is thus concluded that at least two neurons per hemisphere innervate the ventral cord. There are 20 hugin neurons, of which there are four pharyngeal and four ring gland neurons. It is likely, then, that the number of ventral cord neurons is four and that the number of protocerebral neurons is eight (Bader, 2007).

Single hugin cells projecting to the contralateral side of the ring gland were also observed. In addition, these cells are characterized by an ipsilateral process that stops lateral to the esophageal foramen. The neurite length observed can vary. Fibers are consistently observed projecting to the border of the antennal lobe and the SOG; however, the processes sometimes extend farther dorsally. This may be due to thinning of fibers in the more dorsal regions. In the ring gland, the hugin cells establish dense arborizations on the side ipsilateral to the entering fiber and weaker arborizations after crossing to the other side. Possible target cells in the ring gland are located in the corpora cardiaca (Bader, 2007).

The fourth class of hugin neurons projects to the anterior pharynx, close to the cephalopharyngeal skeleton. The neurites leave the SOG, make a U-turn, and end at the anterior part of the dorsal pharyngeal muscles. Whether the pharyngeal neurons in fact innervate the muscles is not known. Neurites can be seen that cross the midline and those that do not, but, because the pharyngeal neurons are located close to the midline, this distinction is sometimes difficult. In addition, these neurons have short neurites along each side of the foramen (Bader, 2007).

The close intermingling of axon endings of gustatory receptor neurons and arborizations of hugin neurons in the SOG suggested that hugin neurons could act as gustatory interneurons. One of the questions raised by the earlier study was to which class of hugin neurons these arborizations belonged. The current analysis suggests that all hugin neurons may have taste inputs; however, neurons that project to the protocerebrum are the only ones showing arborizations in the lateral part of the SOG, whereas other classes of neurons have distinct but overlapping neurites in the medial part of the SOG. Because the SOG lies in close proximity with the antennal lobes, the morphological relationship between the protocerebral hugin neurons and the antennal lobes, the first relay center for olfactory signaling, was investigated. It was first asked whether the arborizations of the protocerebral neurons overlapped with the antennal lobe. Protocerebrum-specific hugin clones in nc82 neuropile background staining indicated that the arborizations lie just at the border of the antennal lobe but do not intermingle with it. This is supported by clones in the adult, where the antennal lobes are significantly larger relative to the SOG. Next, the neurites to the larval protocerebum were analyzed with respect to the mushroom body calyx, a secondary olfactory relay center. Consistent with earlier results, the hugin neurites lie dorsal to the mushroom body calyx. Thus, at the morphological level, no overlap of hugin neurons with the central olfactory pathway is seen. However, insofar as hugin encodes a secreted peptide, an influence on the olfactory system cannot be excluded (Bader, 2007).

The arborizations of the protocerebral hugin neurons lie in a region lateral to the foramen that border the SOG. Other classes of hugin neurons do not show such arborization, but they all show neurites into a region directly juxtaposed to the foramen and the SOG. The architecture of the hugin neurons within this region indicates that the neurites lie in close proximity to each other. The ventral nerve cord neurons, the ring gland neurons, and the pharyngeal neurons all have neurites that extend just lateral to the foramen. For the ventral cord neuron, there is also a small arborization at the bottom end of the foramen, i.e., at the border to the SOG. For the pharyngeal neuron, two additional spiked neurites can be seen extending dorsally and ventrally in a similar region. Thus, in addition to having specific targets outside the SOG, the different hugin neurons have overlapping neurites near the SOG. These observations suggest that the region bordering the lateral foramen and the SOG might have a special role in mediating hugin neuronal function (Bader, 2007).

EFFECTS OF MUTATION

Ectopic hugin expression reduces viability and causes larval death at ecdysis

Out of several tested GAL4-drivers, two caused a marked reduction in the number of eclosed adults. These lines expressed GAL4 either under the ubiquitous tubulin or under a neuroblast-specific promoter. All the flies that had experienced ectopic hugin expression during development, were slightly smaller in size than the controls, otherwise they looked normal. In order to determine the lethal phase, eggs were collected, the number of hatchings was counted, and the development of the larvae was followed until pupariation. No significant differences in the hatching frequencies were noticed between any of the experimental and control crosses. Thus, the embryogenesis seems to proceed normally in the presence of ectopic hugin (Meng, 2002).

During larval development, the highest lethality was observed in larvae with the genotype UAS-hugin 8;tubP-GAL4, which carry two UAS-hugin insertions. Only 5% of the larvae survived until pupariation. The most prominent lethal phase, where 44% of all the larvae died, was around the second larval molt. Quite a few variations were observed between individuals in the precise time of death during the process. Earliest death was observed before the onset of ecdysis, when only duplicated mouth hooks and anterior spiracles were visible. The most advanced larvae had developed two complete cephalopharyngeal skeletons and almost completed ecdysis before dying, partly surrounded by the old cuticle (Meng, 2002).

In addition to the larvae, which simply arrested development, many exhibited defects in morphology and molting behaviour. Two major phenomena were observed: (1) larvae that had shed the old cuticle (partly or completely), but that were still attached to the old cephalopharyngeal skeleton were observed. This phenotype appears to be identical to the buttoned-up phenotype, that could induce it either by deletion of the ETH gene, or by premature injection of Drm-ETH-1. Normally, wild-type larvae always start the ecdysis process by shedding the cephalopharyngeal skeleton. (2) The larvae often developed brown spots on the new cuticle in a regular pattern late during the second instar. The heavily pigmented ones appeared unable to break the old second instar cuticle, although the cuticle with attached spiracles detached from the pigmented third instar cuticle. Instead, the larvae became trapped within a bag of old cuticle, in which they remained alive for some time. Some of these trapped larvae still had not developed a complete new set of the cephalopharyngeal skeleton. This phenomenon constitutes a novel phenotype. Of the larvae that survived through ecdysis, the majority (34% of all hatched larvae) died early during third instar. Taken together, these data suggest that ectopic hugin expression results in induction of premature ecdysis behaviour, which causes the death of the larva (Meng, 2002).

This suggests that at least one peptide encoded by the hugin gene is able to interact with the neuropeptide signalling pathway involved in the ecdysis process. Ecdysteroids are required for the initiation of the events leading to ecdysis, while the ecdysis behaviour itself is regulated by the neuropeptides. In Drosophila, double mouth hooks on larval corpses are considered to be the hallmark of failed ecdysis. Among the described mutants having the double mouthpart phenotype, many have defects in genes involved in ecdysone synthesis. Failed ecdysis is also caused by mutations in genes encoding neuropeptide-processing enzymes. ETH mutants exhibited an abnormal ecdysis behaviour, which often resulted in larvae that only partially managed to escape through an opening in the old cuticle, and which failed to detach their mouth hooks. The same phenomenon was seen in larvae with ectopic hugin expression (Meng, 2002).

Premature induction of ecdysis behaviour was induced by injection of Drm-ETH-1 and results in failed ecdysis, where the animals become trapped in the old cuticle. Hence, the absence as well as the presence of ETH at inappropriate time causes death through premature induction of the ecdysis behaviour. Ectopic hugin could likewise interfere with the ecdysis process, either through induction or inhibition of the ecdysis-signaling cascade. Indeed, the hugγ peptide has primary sequence resemblance to Drm-ETH-1 and could possibly be responsible for premature induction of ecdysis behaviour leading to the death of the larva. It has been experimentally shown that in addition to inducing factors, also inhibiting factors present in the s ganglion play an important role during the ecdysis process, although such an inhibiting factor still remains to be identified. Either of the peptides encoded by hugin could be such a factor. However, further analyses are required to discriminate between the functions and the individual peptides in vivo (Meng, 2002).

A substantial proportion of the larvae died as young third instar larvae. This could be explained with reference to defects that arise during larval development due to prematurely induced but still successful ecdysis, the consequences of which are not fatal until after the molt. Another possibility is that a badly timed myostimulatory effect during normal ecdysis causes problems, which later kill the larva. Abnormal ecdysis can bring along several problems, for example in feeding and respiration, which could be the ultimate cause of death. Death could also be the consequence of cuticular defects. Observations of necrotic patches in the larval cuticula support this option. Less severely affected animals were able to complete ecdysis, but nevertheless died, maybe because the new cuticle did not sustain life. Previously, cuticular defects have been suggested to be the cause of death at the double mouth hook stage in larvae with ectopic FTZ-F1 expression and in larvae mutant for the sas cell surface receptor. A more direct link between cuticular defects and failed ecdysis has been demonstrated in an experiment where a chitin-synthesis inhibitor was fed to the larvae. Finally, it seems that hugin overexpression does not simply operate through the ecdysis control pathway. Eventually, hugin lack-of-function mutants will need to be created and analysed in order to establish the roles of the gene (Meng, 2002).

EVOLUTIONARY HOMOLOGS

Neuromedin U and its putative Drosophila homolog hugin

It is argued that a mammalian homolog of Drosophila hugin may be neuromedin U (NmU). NmU was originally isolated from porcine spinal cord based on its ability to contract uterine smooth muscle. Characterization of porcine NmU identified two peptides with similar bioactivity, a 25-mer (NmU-25) and an 8-mer (NmU-8). NmU-8 is derived from cleavage of NmU-25 and shares an identical C-terminus, which is critical for bioactivity, and is highly conserved among vertebrates (Melcher, 2006).

One of the peptides produced by the Drosophila hugin gene is pyrokinin-2 (PK-2), which also possesses myostimulatory activity. This peptide also bears striking sequence resemblance to mammalian NmU-8. Both are 8-mers, and porcine NmU-8 sequence (YFLFRPRN) and Drosophila PK-2 sequence (SVPFKPRL) share three of eight amino acid residues. The three common residues lie in the last five amino acids; among the vertebrates, the last five residues are identical. Cockroach, Periplaneta americana, pyrokinin sequence (LVPFRPRL) shows even higher homology to porcine NmU-8, with four of eight amino acids being identical, again all in the last five residues. Putative G-protein-coupled receptors for Drosophila PK-2 also share high homology with mammalian NmU receptors (Melcher, 2006).

The structure of the prepropeptides that gives rise to mammalian NmU-8 and Drosophila PK-2 is also similar. Human and rat NmU genes, and hugin, encode prepropeptides that can be potentially cleaved into three peptides. Nmu-8 and Drosophila PK-2 are derived from the last peptide. In Drosophila, the middle peptide was termed hugin-γ. Whether other cleavage products from vertebrates encode functional neuropeptides remains to be determined, but the high conservation between rat and human sequences (Austin, 1995) in this region (36 of 38 identical amino acids) suggests an important function (Melcher, 2006).

Similarities between NmU and hugin extend to the functional level. Rat NmU is specifically expressed in the ventromedial hypothalamus, a region involved in regulating feeding, and its expression is downregulated upon fasting; hugin is specifically expressed in the subesophageal ganglion, a brain region in Drosophila regulating feeding, and its expression is also downregulated upon starvation. Administration of NmU causes suppression of feeding in rats, while NmU knockout in mice causes hyperphagia; in Drosophila, overexpression of hugin causes suppression of growth and feeding, while blocking synaptic activity of hugin neurons causes increased feeding (Melcher, 2006).

REFERENCES

Search PubMed for articles about Drosophila Hugin

Austin, C., Lo, G., Nandha, K. A., Meleagros, L. and Bloom, S. R. (1995) Cloning and characterization of the cDNA encoding the human neuromedin U (NmU) precursor: NmU expression in the human gastrointestinal tract. J Mol Endocrinol 14: 157-169. Medline abstract: 7619205

Bader, R., et al. (2007). Genetic dissection of neural circuit anatomy underlying feeding behavior in Drosophila: distinct classes of hugin-expressing neurons. J. Comp. Neurol. 502(5):848-56. Medline abstract: 17436293

Gendre, N., et al. (2004). Integration of complex larval chemosensory organs into the adult nervous system of Drosophila. Development 131: 83-92. Medline abstract: 14645122

Hückesfeld, S., Peters, M. and Pankratz, M.J. (2016). Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophila brain. Nat Commun 7: 12796. PubMed ID: 27619503

Melcher, C. and Pankratz. M. J. (2005). Candidate gustatory interneurons modulating feeding behavior in the Drosophila brain. PLoS Biol. 3(9):e305. Medline abstract: 16122349

Melcher, C., Bader, R., Walther, S., Simakov, O. and Pankratz, M. J. (2006). Neuromedin U and its putative Drosophila homolog hugin. PLoS Biol. 4(3):e68. Medline abstract: 16524341

Melcher, C., Bader, R. and Pankratz, M. J. (2007). Amino acids, taste circuits, and feeding behavior in Drosophila: towards understanding the psychology of feeding in flies and man. J. Endocrinol. 192(3): 467-72. Medline abstract: 17332516

Meng, X., et al. (2002). The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides. Mech. Dev. 117(1-2): 5-13. Medline abstract: 12204246

Park, Y., Kim, Y. J. and Adams, M. E. (2002). Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc. Natl. Acad. Sci. 99(17): 11423-8. Medline abstract: 12177421

Predel, R., et al. (1999). Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach. Insect Biochem. Mol. Biol. 29: 139-144. Medline abstract: 10196736

Rosenkilde, C., et al. (2003). Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2. BBRC 309: 485-494. Medline abstract: 12951076

Schlegel, P., Texada, M. J., Miroschnikow, A., Schoofs, A., Huckesfeld, S., Peters, M., Schneider-Mizell, C. M., Lacin, H., Li, F., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2016). Synaptic transmission parallels neuromodulation in a central food-intake circuit. Elife 5: e16799. PubMed ID: 27845623

Schoofs, A., Hückesfeld, S., Schlegel, P., Miroschnikow, A., Peters, M., Zeymer, M., Spiess, R., Chiang, A. S. and Pankratz, M. J. (2014). Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain. PLoS Biol 12: e1001893. PubMed ID: 24960360

Wang, Z., Singhvi, A., Kong, P. and Scott, K. (2004). Taste representations in the Drosophila brain. Cell 117: 981-991. Medline abstract: 15210117