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

Synonyms -

Cytological map position- 87B15-87B15

Function - neuropeptides

Keywords - feeding behavior, ecdysis behaviour, brain interneurons

Symbol - Hug

FlyBase ID: FBgn0028374

Genetic map position - 3R

Classification - pyrokinin and ecdysone triggering hormone homolog

Cellular location - secreted



NCBI links: Precomputed BLAST | EntrezGene | UniGene | PubMed articles
Recent literature
Hückesfeld, S., Peters, M. and Pankratz, M.J. (2016). Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophilabrain. Nat Commun 7: 12796. PubMed ID: 27619503
Summary:
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 brain 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.


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).


GENE STRUCTURE

cDNA clone length - 1033 bp

Bases in 5' UTR - 168

Exons - 3

Bases in 3' UTR - 289

PROTEIN STRUCTURE

Amino Acids - 191 (preproprotein)

Structural Domains

Screening of a Drosophila genomic cosmid library at low stringency with a human GDNF cDNA probe followed by subcloning yielded a 1 kb EcoRI genomic fragment containing the most similar area. This fragment was used to probe a Northern blot and screen a cDNA library representing all stages of embryogenesis. A 1 kb-cDNA clone (pXM1-cDNA) was isolated and sequenced. A 4 kb genomic EcoRI-fragment upstream of the original 1 kb genomic fragment was also isolated and sequenced, allowing for the analysis of the gene structure. A canonical TATA-box and CAP-site were identified on the genomic sequence within 50 nucleotides upstream of the cDNA start. This, together with the size of the transcript as identified on the Northern blot, were taken as bona fide indication that the cDNA was a full-length clone. The fly gene was named hugin and the sequence data entered into the EMBL database with the accession number AJ133105. The sequence analysis of the cDNA revealed the presence of a 573 bp coding sequence (CDS). The conceptual translation yields a 191-residue polypeptide with a predicted molecular weight of about 21 kDa with a signal sequence (amino acids 1–24) (Meng, 2002).

Western blot analysis of conditioned media from S2 cells transfected with the fusion construct pMThugV5H6 using anti-V5 antibody revealed a pattern of small molecular size polypeptides, with the major bands having a molecular weight of about 5 and 8 kDa. Also, larger bands of about 18 and 23 kDa were visible. To identify the fragments, the C-terminally His-tagged secretion products were purified from the media and the strongest bands were isolated for N-terminal amino acid sequencing. The sequences obtained were SIDSWRLL and SVPFKPR, positioning the cleavage sites to the N-terminal side of serines 140 and 174. The calculated molecular weights of the proteolytic fragments including the C-terminal tags were 8.3 and 4.7 kDa, respectively, corresponding to the 8 and 5 kDa bands in the gel. The fainter 22 kDa band probably represents a small amount of unprocessed fusion protein without signal sequence (calculated weight 20.9 kDa). Also, a minor band of apparent weight of 18 kDa was visible, positioning the N-terminus around 30–40 amino acids after the signal sequence cleavage site. Under non-reducing conditions multiple bands ranging in size between 8 and 43 kDa were seen, most likely representing the various hetero- and homodimers resulting from disulphide pairing of the processing intermediates via the cysteine at position 188 (Meng, 2002).

Clusters of basic residues, which could act as targets for Drosophila pro-protein and pro-hormone convertases were found preceding the confirmed processing sites. Three additional putative convertase target sites, promoting cleavage after arginine residues 62, 120 and lysine 184, were identified. Processing at R62 would result in a 17.1 kDa C-terminal cleavage product, which could be responsible for the 18 kDa band in the Western blot. Cleavage at R120 would create an intermediate peptide, which would be converted to an amidated form QLQSNGEPAYRVRTPRL-NH2 (hugγ) by the successive actions of carboxypeptidase E (encoded by silver), peptidylglycine-α-hydroxylating mono-oxygenase (PHM) and peptidyl α-hydroxyglycine-α-amidating lyase (PAL). Another amidated peptide SVPFKPRL-NH2 would result by cleavage at the sites K173 and R184 (Meng, 2002).

The obviously convertase-dependent cleavage of the hugin precursor was restricted to the S2 cell line, indicating a specificity of hugin processing. Overexpression of hugin in COS7 cells resulted in a major secretion product of the predicted size of full-length hugin after signal sequence removal. The lack of processing in COS7 cells either reflects the low amounts of furins in this cell line or the inability of mammalian convertases to recognise the processing motifs in the hugin precursor (Meng, 2002).

One of the hugin peptides, SVPFKPRL-NH2 , was found to be similar to the recently identified pyrokinin 3 (Pea-PK-3) from the American cockroach, P. americana (Predel, 1999). The Drosophila peptide contains the FXPRL-amide structure common to all known pyrokinins and was named Drm-PK-2. The longer peptide, tentatively named hugγ has the same C-terminal PRL-amide designation, but lacks the preceding phenylalanine. The highest similarity scores were obtained with another Drosophila peptide, the ecdysis-triggering hormone 1 (Drm-ETH-1) encoded by the gene ETH (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).


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

date revised: 15 June 2007

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