InteractiveFly: GeneBrief

Diuretic hormone 44: Biological Overview | References

Gene name - Diuretic hormone 44

Synonyms -

Cytological map position - 85E2-85E2

Function - hormone

Keywords - brain neurosecretory cells, hormone, sensor of nutritive sugar, directs the detection and consumption of nutritive sugar, leads to proboscis extensions and frequent episodes of excretion, control of sperm retention and storage by females, circadian output hormone that is required for normal rest:activity rhythms

Symbol - Dh44

FlyBase ID: FBgn0012344

Genetic map position - chr3R:9,638,182-9,650,110

Classification - corticotropin-releasing factor

Cellular location - secreted

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
King, A. N., Barber, A. F., Smith, A. E., Dreyer, A. P., Sitaraman, D., Nitabach, M. N., Cavanaugh, D. J. and Sehgal, A. (2017). A peptidergic circuit links the circadian clock to locomotor activity. Curr Biol 27(13): 1915-1927 e1915. PubMed ID: 28669757
The mechanisms by which clock neurons in the Drosophila brain confer an approximately 24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. This study identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and its site of action was mapped to hugin-expressing neurons in the subesophageal zone (SEZ). A circuit was traced that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.
Zandawala, M., Marley, R., Davies, S. A. and Nassel, D. R. (2017). Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila. Cell Mol Life Sci [Epub ahead of print]. PubMed ID: 29043393
Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. This study investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. It was also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. These data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.

Animals can detect and consume nutritive sugars without the influence of taste. However, the identity of the taste-independent nutrient sensor and the mechanism by which animals respond to the nutritional value of sugar are unclear. This study reports that six neurosecretory cells in the Drosophila brain that produce Diuretic hormone 44 (Dh44), a homolog of the mammalian corticotropin-releasing hormone (CRH), are specifically activated by nutritive sugars. Flies in which the activity of these neurons or the expression of Dh44 was disrupted failed to select nutritive sugars. Manipulation of the function of Dh44 receptors had a similar effect. Notably, artificial activation of Dh44 receptor-1 neurons resulted in proboscis extensions and frequent episodes of excretion. Conversely, reduced Dh44 activity led to decreased excretion. Together, these actions facilitate ingestion and digestion of nutritive foods. It is proposed that the Dh44 system directs the detection and consumption of nutritive sugars through a positive feedback loop (Dus, 2015).

Sugars in the natural environment can be detected through taste-dependent and taste-independent modalities. Taste-dependent modalities consist mainly of peripheral taste receptor cells such as sweet-sensing cells, which primarily detect the palatability of sugar. Evidence of a taste-independent modality was shown more than 20 years ago when investigators showed that rodents could learn to select a flavored solution when it was paired with an intragastric infusion of nutritive sugars but not with water or nonnutritive saccharin. This finding was further demonstrated by experiments using taste-insensitive Trpm5 (-/-) mice, which learn to associate nutritive sugars paired with a conditioned stimulus independent of taste input. Similarly, fruit flies-Drosophila melanogaster-are capable of associating the caloric value of sugars with an odorant to establish a long-term memory (Dus, 2015).

While animals and humans can learn to recognize the nutritional value of sugar during sugar-preference conditioning, Drosophila do not need to be trained to distinguish between nutritive sugars and nonnutritive sugars. Studies have shown that naive flies that had not previously been exposed to nutritive sugars or nonnutritive sugars were still able to select nutritive sugars over nonnutritive ones after periods of food deprivation in a two-choice preference assay. The post-ingestive preference for a nutritive sugar appears to be mediated by a hardwired neuronal pathway that is activated by the detection of nutritive sugars. However, the molecular and cellular identity of the nutrient sensor and the neural circuitry that allows flies (as well as mammals) to respond to the nutritional value of exogenous sugar is largely unknown (Dus, 2015).

The postprandial increase in the intestinal and circulating glucose levels plays an important role in the ability of animals to choose conditioned stimuli paired with nutritive sugars. Several studies in rodents showed that intravenous glucose administration is sufficient for preference conditioning, while direct stimulation of the intestinal mucosa was also shown to be important. This relationship was further supported by the observation in flies that administrating phlorizin, which lowers hemolymph glycemia by inhibiting sugar transport, blocked the flies' ability to select nutritive sugars. Notably, taste-independent sugar conditioning was shown to correlate with the rate of glucose utilization instead of circulating glucose levels in mice. In humans, the physiological parameter that appears to correlate with preference conditioning is also metabolic responses to glucose. While these studies illustrate that utilizing intracellular glucose is crucial for activating behavioral responses, circulating plasma glucose level is key in determining intracellular glucose concentration (Dus, 2015).

Indeed, Jean Mayer proposed over 5 decades ago that feeding is regulated by neurons in the brain that sense circulating blood glucose levels. This 'glucostatic hypothesis' was substantiated by the discovery of glucose-sensing neurons in the hypothalamus. These specialized neurons use the products of glucose metabolism to regulate neuronal excitability and neurotransmitter release. Metabolic enzymes such as glucokinase, the AMP-activated protein kinase (AMPK), and the ATP-sensitive K+ (KATP) channel were implicated in mediating this process. However, the disruption of KATP channel or AMPK function in glucose-excited pro-opiomelanocortin (POMC) neurons, which impaired their ability to sense glucose, did not result in a discernable feeding phenotype in mice. While several populations of glucose-sensing neurons have been identified in the hindbrain and hypothalamus, their biological role in feeding-related behavior is still elusive (Dus, 2015).

This work has identified a population of neurons in the fly brain producing the Diuretic hormone 44 neuropeptide (Dh44, the insect homolog of the mammalian CRH) (Lovejoy, 2006) that is essential for mediating taste-independent behavioral responses to the nutritional value of sugar. Calcium imaging revealed that Dh44 neurons are activated by solutions containing nutritive sugars and require a functional glucokinase enzyme to detect these sugars. The Dh44 neuropeptide conveys the information from Dh44 neurons to Dh44 receptor R1 neurons in the brain and R2 cells in the gut, both of which are also required for nutrient selection. Furthermore, artificial activation of Dh44 R1 neurons stimulated rapid proboscis extension reflex (PER) responses, promoting food intake. Flies with activated Dh44 R1 neurons also excreted more frequently, a behavior likely increased by gut motility. Conversely, reduced Dh44 signaling resulted in a lower frequency of excretion. It is proposed that this putative post-ingestive nutrient sensor activates two pathways: one to promote PER to reinforce the ingestion of nutritive foods and another to enhance the gut motility, which would facilitate digestion of greater volumes of the nutritive foods (Dus, 2015).

This study has identified the molecular and cellular nature of a sensor in the brain that detects the nutritional value of sugar through direct activation by nutritive sugars. Dh44 neurons are activated specifically by nutritive D-glucose, D-trehalose, and D-fructose, which are normally found in the hemolymph, and are not activated by nonnutritive sugars or sugars that are not found in the hemolymph. Sugar-induced activation of these six central neurons resulted in secretion of the Dh44 neuropeptide, which transmits a signal to Dh44 R1 and R2 cells. Flies in which the expression of Dh44 or Dh44 receptors is disrupted or the function of Dh44 receptor cells is inactivated failed to develop a preference for nutritive sugar (Dus, 2015).

Insight into the contribution of the Dh44 downstream effectors to the selection of nutritive sugars was gained in the TrpA1-mediated activation experiment. The surprising observation was made that artificial activation of Dh44 R1 neurons rapidly induced PER responses, even in the absence of food. Stimulation of Dh44 R1 neurons also caused the flies to excrete large amounts of waste deposits; conversely, inactivation of the Dh44 circuit resulted in deceleration of gut motility and excretion. Together, it is proposed that the Dh44 system not only mediates detection of the nutritional content of sugar but also coordinates the ingestion and digestion of sugar by promoting proboscis extension and by promoting gut motility and excretion through a positive feedback loop (Dus, 2015).

Two possible mechanisms could explain how flies can make appropriate food choices in the two-choice assay. One mechanism is regulated by a post-ingestive nutrient sensor that detects the nutritional value of D-glucose through direct activation during the postprandial rise in hemolymph glycemia. Another mechanism is mediated by a prescriptive 'hunger' sensor that monitors the status of the internal energy reservoir and promotes consumption of nutritive D-glucose after periods of starvation (Dus, 2015).

Several lines of evidence suggest that Dh44 neurons function as a post-ingestive nutrient sensor. First, Dh44 neurons are activated specifically by nutritive sugars and not by nonnutritive sugars. Second, Dh44 neurons are capable of directly sensing the nutritional value of sugar, as sugar-induced calcium responses were not eliminated in fly brains treated with TTX, a sodium channel blocker that abolishes synaptic transmission. Third, flies with Hex-C knocked down in these Dh44 neurons had impaired responses to nutritive sugar. Fourth, artificial activation of Dh44 neurons or Dh44 R1 neurons significantly reduced the preference for nutritive sugars, even when the flies were starved, because activation of the putative nutrient-sensing pathway was sufficient to communicate the reward of nutrient. Therefore, starved flies carrying PDh44-GAL4 and UAS-NachBac equally preferred D- and L-glucose. This is in contrast to another population of central neurons identified from a screen that functions as a prescriptive hunger sensor. When these neurons were artificially activated, the flies chose a nutritive sugar over a nonnutritive sugar, even when they were sated. Finally, either activation or inactivation of Dh44 neurons did not alter the amount of food consumption. This is distinct from manipulating the prescriptive hunger sensor that had substantial effects on the amount of food intake. These support the assertion that the glucose-sensing Dh44 neurons guide flies to recognize the nutritional value of sugar by directly monitoring circulating sugar levels and utilizing sugar molecules (Dus, 2015).

The means by which flies distinguish D-glucose from L-glucose are not understood. It was proposed that flies roaming in the two-choice arena find D-glucose by associating a spatial cue, the location of the D-glucose containing agar, with the nutritional content of D-glucose. The observation that these flies are capable of selecting D-glucose even in the dark, however, suggests that spatial conditioning is unlikely to be involved in post-ingestive food choice behavior. Rather, the detection of nutritive D-glucose appears to be mediated by interoceptive chemosensory neurons that elicits innate behavioral responses, similar to the sweet-evoked chemosensory responses mediated by external sweet receptors. Upon activation by a nutritive sugar, the interoceptive chemosensory neurons stimulate a constellation of behavioral sub-programs that result in a positive feedback for the selection and consumption of nutritive sugar (Dus, 2015).

Consistent with this hypothesis, the post-ingestive nutrient sensor functions in a fast timescale. Calcium imaging of dissected ex vivo brain preparations, which may not reflect the in vivo context in which ingested foods pass through the digestive tract, showed that the activity of Dh44 neurons is rapidly stimulated when exposed to nutritive sugar. Furthermore, hemolymph glycemia significantly increases as soon as flies start to ingest sugars. The rise of hemolymph glycemia would readily stimulate the activity of Dh44 neurons, which are located adjacent to insulin-producing cells (IPCs) in the PI that also respond to sugar. Finally, the time-course experiment demonstrated that flies that begin to feed in the two-choice assay are capable of responding correctly to nutritive D-glucose within 5 min. These results support the view that flies recognize the nutritional content of D-glucose rapidly after ingestion (Dus, 2015).

It has been 5 decades since the glucostatic hypothesis was proposed, yet it is still uncertain whether glucose-sensing neurons in the brain have a role in food intake or nutrient selection. Mice that lack a critical signal transducer, either AMPK or KATPchannel, in their glucose-sensing neurons and, thus, lack the ability to sense extracellular glucose display essentially normal feeding behavior. A study in rats also showed a lack of any causal relationship between blood and hypothalamic glucose levels and daily meal initiations. Recently, hypothalamic glucose-sensing melanin-concentrating hormone (MCH) neurons were shown to respond to and communicate the nutritional value and reward of sugar, but it was not clear whether the glucose excitability of these MCH neurons mediated the behavioral response. However, central administration of 2-deoxyglucose or insulin-induced hypoglycemia does elicit food intake. It was speculated that extremely low brain glucose levels trigger food intake through the action of unidentified hypothalamic glucose-sensing neurons, which may protect against the dangers of hypoglycemia in mammals (Dus, 2015).

This study in Drosophila has shown that the glucose excitability of Dh44 neurons mediates starvation-induced selection of nutritive sugars, which depends on the sugar entry and the function of Hex-C to convert the glucose into its metabolic product, glucose-6-phosphate. This step in the glucose metabolic pathway appears to be critical for stimulating the neuronal activity in Dh44 neurons and responding to the nutritional value of sugar. It is noteworthy that Hex-C mRNA is expressed in few regions, including the brain, whereas another fly hexokinase, hexokinase A (Hex-A), is expressed in nearly all tissues in the fly. The intracellular glucose metabolism initiated by Hex-C, possibly through the generation of sugar metabolites, is important for detecting the nutritional value of D-glucose that elicits innate preference behavior (Dus, 2015).

Since it was discovered 25 years ago, CRH has been characterized as a hypothalamic hormone that communicates stress responses. CRH also plays a significant role in the regulation of energy balance, but the exact nature of its role is controversial. CRH appears to have an anorectic effect in rodents but has an opposite effect in humans when calorie intake is stimulated by an infusion of CRH. The homology between Drosophila Dh44 and mammalian CRH is approximately 30% and between Drosophila and mammalian receptors is approximately 40%; this suggests that the function of these two systems is conserved. Indeed, mammalian CRH, which is similar in function to Drosophila Dh44, is required for the regulation of gastric and colonic movements; notably, CRH administration was shown to stimulate defecation in rodents. Furthermore, CRH mediates glucose homeostasis by regulating hypoglycemia-induced counterregulation (CRR). CRR triggers a number of responses that limit glucose utilization, promote endogenous glucose production, and lead the animal to seek food. It has been suggested that the function of glucose-sensing neurons is to generate neuroendocrine stress responses to the hypoglycemic challenge, but the identity of these neurons is unknown. It would be interesting to investigate the possibility that CRH neurons, which are expressed in the hypothalamus, are glucose-sensing neurons and capable of mediating starvation-induced behavioral responses to the nutritional value of sugar in mammals. A stress-responsive CRH system might be co-opted to allow animals to respond to the stress of starvation (Dus, 2015).

A neuronal pathway that controls sperm ejection and storage in female Drosophila

In polyandrous females, sperm storage permits competition between sperm of different mates, and in some species females influence the relative fertilization success of competing sperm in favor of a preferred mate. In female Drosophila melanogaster, sperm competition is strongly influenced by the timing of sperm ejection from the uterus. Understanding how female behavior influences sperm competition requires knowledge of the neuronal mechanisms controlling sperm retention and storage, which is currently lacking. This study shows that D. melanogaster females eject male ejaculates from the uterus 1-6 hr after mating with a stereotypic behavior regulated by a brain signaling pathway composed of Diuretic hormone 44 (Dh44), a neuropeptide related to vertebrate corticotropin-releasing factor (CRF), and its receptor, Dh44R1. Suppression of Dh44 signals in the brain expedited sperm ejection from the uterus, resulting in marked reduction of sperm in the storage organs and decreased fecundity, whereas enhancement of Dh44 signals delayed sperm expulsion. The Dh44 function was mapped to six neurons located in the pars intercerebralis of the brain together with a small subset of Dh44R1 neurons that expressed the sex-specific transcription factor doublesex. This study identifies a neuronal pathway by which females can control sperm retention and storage and provides new insight into how the female might exercise post-copulatory sexual selection (Lee, 2015).

Male D. melanogaster avoid sperm competition by discouraging females from re-mating and by depositing a mating plug in the female uterus. The mating plug is presumed to block insemination by another male and retain sperm in the uterus, allowing time for sperm storage. In mated D. melanogaster females, sperm collects in the uterus at the openings to the sperm storage organs, comprising seminal receptacle (SR) and paired spermathecae (Sp). However, as little as 10%-20% of the uterine sperm is stored, with most of the ejaculate being lost at the same time as ejection of the gelatinous plug. Despite the potential for the mating plug to influence the efficiency of sperm storage and sperm competition, nothing is known about plug ejection behavior and how this is regulated by the female. Thus, the behavior of D. melanogaster females in actively removing sperm and the mating plug from a first mating was studied by video recording individual mated females. At various times after the end of copulation (AEC), females groomed extensively and squeezed from the vagina a white gelatinous sac comprising both the sperm mass and the mating plug. Henceforth, this behavior is referred to as sperm ejection. When wild-type Canton S (CS) and the eye-color mutant w1118 flies were examined, it was found that sperm ejection occurred at times that varied widely within and between genotypes. Next, protamine-GFP males, the sperm heads of which express GFP, were used to examine the dynamics of sperm retention in the uterus at various times after mating. Immediately after mating, females had a large sperm mass in the uterus together with the autofluorescing mating plug . When examined at 60 min AEC, the sperm mass and mating plug were still present in the uterus, and sperm had appeared in the SR. At 180 min AEC, a large proportion of mated females had high numbers of sperm stored in the SR and Sp, but both sperm and the plug were absent from the uterus. Notably, the ejected sperm mass was always observed together with the mating plug, linking the ejection of un-stored sperm with the removal of the mating plug. Thus, post-mated D. melanogaster females actively remove un-stored sperm alongside the mating plug at variable times AEC (Lee, 2015).

It was speculated that this behavior is regulated by a central peptidergic pathway since peptides have been implicated in many female reproductive behaviors. To identify peptidergic pathways that modulate sperm ejection, a comprehensive panel of neuropeptide genes was knocked down using the pan-neuronal nSyb-Gal4 driver, and the timing of sperm ejection was measured in post-mated females. This small RNAi screen identified diuretic hormone 44 (Dh44), knockdown of which resulted in precocious ejection of both sperm and the mating plug from the uterus of post-mated females within 10 min AEC. In contrast, females lacking the RNAi transgene or carrying other neuropeptide-RNAi transgenes ejected sperm after much longer delays, >1 hr. At 1 hr AEC, post-mated Dh44-RNAi females had lost the mating plug and held little or no sperm anywhere in the entire reproductive system, whereas control females retained both the plug and uterine sperm at the same time as sperm was stored in the SR. To control potential off-target effects, three different Dh44-RNAi lines were prepared, and all three were confirmed to display precocious sperm ejection with similar reduced latency. Further examination of Dh44-RNAi females 3 and 6 hr AEC confirmed either the complete absence or severe reduction of the number of sperm in the SR. To rule out the possibility that Dh44 is involved in the initial sperm transfer from males, copulation duration was measured and sperm in the uterus were counted immediately AEC, and no difference was found between Dh44-RNAi and control females (Lee, 2015).

Upon mating, Drosophila females display an elevated rate of egg laying and mating refractoriness, known collectively as the post-mating responses (PMRs). These can be divided into short-term (12-24 hr) and long-term (6-7 days) phases elicited primarily by the seminal fluid proteins (SFPs) DUP99B and SP. SP is delivered to females attached to sperm, and it is the slow release of this peptide from stored sperm that elicits the long-term PMR. It was therefore expected that precocious sperm ejection and the consequential reduction of stored sperm would prevent post-mated Dh44-RNAi females from displaying normal long-term PMRs. Indeed, compared to control flies, Dh44-RNAi females laid 50%-80% fewer eggs over a 48 hr period, all of which developed to adults. Mating refractoriness usually lasts about 1 week after mating, whereas females that mated with SP-less males resist mating only until 12 hr after the first mating. This short-term change in behavior is probably a response to DUP99B, which, although structurally related to SP, is not carried by sperm.The relationship between sperm ejection and PMR development was further investigated by examining mating refractoriness of Dh44-RNAi females at various times after the first mating. Then, they were compared with CS females mated with either SP-null or CS males. As expected, CS females mated with wild-type males remain refractory to further mating at 48 hr AEC, whereas CS females mated with SP-less males (SP0/Δ130) stop being refractory from 24 hr AEC, displaying 60%-70% re-mating frequencies. In stark contrast, Dh44-RNAi females displayed significantly higher re-mating frequency even 4 hr AEC and more than 40% re-mating frequencies at 12 hr AEC. These results suggest that precocious sperm ejection caused by Dh44-RNAi prevents females from developing both short- and long-term PMRs as a result of the failure to hold the ejaculate in the uterus for sufficient time for DUP99B and the sperm-tethered SP to elicit a female PMR (Lee, 2015).

As a first step to map Dh44 neurons in the CNS, Dh44 mRNA expression was examined in the adult CNS using in situ hybridization. Dh44 expression in the CNS was broad and complex, occurring in large numbers of cells in the brain and the ventral nerve cord (VNC). In contrast, no cells were stained with the control probe against antisense sequence of Dh44 mRNA. Among the Dh44 cells in the CNS, it was noted that six cells located in the pars intercerebralis (PI) of the brain (Dh44-PI) displayed the most prominent Dh44 mRNA expression. Notably, these six Dh44-PI neurons were recently implicated in circadian control of rhythmic locomotor activity (Lee, 2015).

To further characterize Dh44 neurons, Dh44-Gal4 was generated, and it was found that knockdown of Dh44 in Dh44-Gal4 neurons (Dh44>Dh44-RNAi) causes precocious sperm ejection, similar to that seen with pan-neural Dh44-RNAi. Subsequently, membrane-tethered EGFP was expressed using Dh44-Gal4 (Dh44>mCD8-EGFP) to show that Dh44-Gal4 had a broad expression pattern. Dh44-Gal4 expression was most intense in PI neurons, matching the localization of Dh44 mRNA. The CNS of Dh44>mCD8-EGFP females was stained with both anti-Dh44 and anti-EGFP, and it was found that Dh44-PI and other Dh44-Gal4 neurons are indeed positive for Dh44. The Dh44 immunoreactivity was abolished or greatly attenuated in Dh44-RNAi neurons, verifying the specificity of the antibody staining and the efficacy of the RNAi. Nevertheless, significant numbers of Dh44-Gal4 neurons lacking Dh44 immunoreactivity were noted and vice versa. The female reproductive organs were examined for the expression of Dh44 mRNA, Dh44 immunoreactivity, and Dh44-Gal4 activity, but no evidence was found for Dh44 expression in these tissues (Lee, 2015).

Since many central neurons express Dh44-Gal4, Gal4 activity was restricted by including Gal80 transgenes that suppress transcriptional activity of Gal4 in defined populations of neurons. Acetylcholine is one of major neurotransmitters in the invertebrate CNS, and choline acetyltransferase (cha) encodes an enzyme responsible for the synthesis of acetylcholine. Inclusion of cha-Gal80 was found to suppress Gal4 activity in almost all Dh44-Gal4 neurons, except for six Dh44-PI and several small neurons, allowing examination of anatomy of Dh44-PI neurons at high resolution. Somata of the six Dh44-PI neurons are located in the medial region of the PI. They send major processes along the anterior midline of the brain, arborizing mainly in the prow, which corresponds to the anterior dorsal area of the subesophogeal zone (SOZ). The subsequent RNAi experiment revealed that normal sperm ejection requires Dh44 expression in the Dh44-Gal4 minus cha-Gal80 neuron population, implicating the Dh44-PI neurons in the control of sperm ejection (Lee, 2015).

Salt-induced kinase 2 (Sik2) has been implicated in transcriptional regulation of corticotropin-releasing factor (CRF), a mammalian ortholog of Dh44, in the rat hypothalamus. Thus, it was hypothesized that Drosophila Sik2 is also expressed in Dh44-PI neurons. Indeed, Sik2-Gal4 activity was colocalized with Dh44 exclusively in the six Dh44-PI cells, and Sik2-Gal4-driven Dh44-RNAi also resulted in the precocious sperm ejection, recapitulating Dh44>Dh44-RNAi (Lee, 2015).

Two related G-protein-coupled receptors, Dh44R1 and Dh44R2, have been identified as receptors for Dh44. As seen with Dh44-RNAi, Dh44R1-RNAi in the nervous system resulted in precocious sperm ejectionand a marked reduction in sperm storage. In contrast, three Dh44R2-RNAi lines showed no sign of early sperm ejection and defective storage, although Dh44R2 expression is clearly reduced. Then, Dh44R1-Gal4 was generated, and it was found that knockdown of Dh44R1 in Dh44R1-Gal4 neurons also caused precocious sperm ejection and a reduction in stored sperm. Like its ligand, Dh44R1 showed a broad and complex expression pattern in the CNS. Next, it as asked whether Dh44R1-Gal4 neurons that express fruitless (fru) or doublesex (dsx) regulate sperm ejection because these sexually dimorphic transcription factors have been causally linked to other gender specific behaviors. Dh44R1 neurons positive for fru or dsx were genetically targeted by combining Dh44R1-Gal4 with fruFLP or dsxFLP. Using these flies, it was asked whether depolarization of either subset with a warmth-activated cation channel (dTrpA1) would delay sperm ejection. Thermal activation of dsx-positive Dh44R1 neurons resulted in a marked delay in sperm ejection, but activation of fru-positive ones did not. Consistent with this observation, thermal activation of Dh44 neurons also delayed sperm ejection to a similar extent. RNAi-knockdown of Dh44R1 was performed in either fruGal4 or dsxGal4 neurons. It was not possible to examine sperm ejection of dsxGal4-driven Dh44R1-RNAi females because they resisted mating, whereas fruGal4-driven Dh44R1-RNAi showed no effect on sperm ejection. Subsequently, mCD8-EGFP was expressed in the dsx-positive Dh44R1 neurons and a small number of pairs of neurons were found to be labeled in the female brain. The EGFP labeling also occurred in several neurons of the abdominal ganglia (AG), but not the reproductive organs. Noticeably, dsx-Dh44 neurons displayed marked sex differences in the numbers of soma and in the brain projection patterns, suggesting their gender-specific functions. Dh44R1 mRNA expression in the brain and VNC was examined using in situ hybridization, and its expression pattern was similar to Dh44R1-Gal4 expression, particularly in AG neurons. Brain dsx-Dh44R1 neurons project to distinct brain regions including the PI, but not into the SOZ, to where Dh44-PI mainly project. This suggests that Dh44 may act on these receptor neurons via volume transmission, a common neuropeptide mode of action. It is also conceivable that Dh44 from PI neurons acts on AG neurons, probably through an endocrine route (Lee, 2015).

This study provides compelling evidence that a stereotypic plug/sperm-ejection behavior of post-mated D. melanogaster females is regulated by a central peptidergic pathway composed of Dh44 and Dh44R1 and that early removal of the uterine sperm hinders transfer of sperm to the female sperm storage organs. The evidence does not formally establish that the Dh44 pathway directly regulates sperm ejection. However, it does uncover a pathway, the activity of which can modulate the timing of sperm ejection with high activity resulting in a longer delay to sperm ejection. This is potentially significant in the context of post-copulatory sexual selection since it has been shown that the timing of plug and sperm ejection from the female has a direct effect on the fertilization success of sperm from competing males. Thus, future studies are warranted to link the Dh44 pathway directly to the post-copulatory sexual selection by the female (Lee, 2015).

The reproductive success of males will often depend upon avoidance of sperm competition by discouraging females from re-mating. In D. melanogaster, SFPs are known to increase male fitness by manipulating female physiology and behavior (female PMR) and by forming a mating plug. By suppressing Dh44 signaling in the brain, it was possible to weaken female PMRs. This raises the intriguing possibility that D. melanogaster females may be able to counter the behavior-modifying SFPs through the Dh44 pathway (Lee, 2015).

Dh44 and Dh44R1 are orthologous to CRF and CRFR, important coordinators of the physiological response to stress in vertebrates. Furthermore, the Dh44 function mapped to six PI neurons expressing Sik2, a gene implicated in stress responses and transcription of CRF, suggesting the possible evolutionary conservation of this signaling pathway at several levels. The physical act of copulation in addition to the transfer of toxic SFPs in the ejaculate can compromise Drosophila female fitness. Thus, it is plausible that female flies activate the stress pathway to cope defensively with mating-associated insults and costs and that this pathway has evolved new functions associated with retention of the ejaculate and sperm storage. It is therefore of interest to ask whether CRF mediates copulation-associated physiological responses in vertebrates (Lee, 2015).

What signals in the female brain might regulate the Dh44 pathway? It is noted that Dh44-PI neurons occur in both males and females, suggesting additional functions common to both sexes. Cavanaugh (2014) reported that Dh44-PI form part of circadian output circuits, suggesting functional pleiotropy of this pathway. Likewise, CRF peptides in mammals have been implicated in diverse processes, such as the timing of parturition, feeding, vascular tone, and cardiac functions. Dh44-PI neurons may also have evolved as a brain center for integrating multiple signals from circadian, metabolic, sensory, and reproductive systems for coordinated modulation of sperm ejection and other behaviors. This identification of a circuit that regulates timing of sperm ejection is a critical step forward in understanding how females might modulate post-copulatory responses by integrating external signals such as the quality and quantity of the ejaculate and the social and physical environments with internal physiological signals associated with nutrition, reproduction, and circadian rhythms (Lee, 2015).

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).

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. This study investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. It was also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. These data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles (Zandawala, 2017).

This study reveals that a portion of the LK-expressing neurosecretory cells (ABLKs) in abdominal ganglia co-express DH44, similar to earlier findings in the moth Manduca sexta, the locust Locusta migratoria, and blood-sucking bug Rhodnius prolixus. Colocalization of these peptides in multiple insect orders, including basal orders, suggests that this colocalization and the subsequent functional interaction between these signaling systems evolved early on during insect evolution. Since ABLKs are the sole neurons producing both peptides in Drosophila, it was possible to use GAL4 lines to knock down each of the two peptides in these cells only and thereby isolate the contribution of the ABLKs to the physiology. This enabled establishing that these neuroendocrine cells are sufficient for many of the functions assigned to DH44 and LK and therefore these functions are hormonally mediated. In contrast, earlier studies were based upon altering peptide levels or activity in entire populations of DH44 and LK neurons. Also, this study showed that the LK-GAL4 driver includes a set of ectopic brain cells (ipc-1) that do not express LK, but another peptide ITP. The ipc-1 neurons produce sNPF and tachykinin in addition to ITP and have been found to regulate stress responses. This means that in earlier studies, where the LK-GAL4 line was used to inactivate or activate neurons, additional phenotypes are likely to have arisen. Using the current approach, only ABLK neurons were targetted, it was found that both DH44-RNAi and Lk-RNAi in these cells increase resistance to desiccation, starvation, and ionic stress. This suggests that diminishing the release of these two peptides from ABLKs is sufficient for this phenotype to occur. However, food intake is not affected by LK knockdown in ABLKs, whereas DH44 knockdown diminishes feeding, and conversely the knockdown of LK in ABLKs results in increased body water content that is not seen after DH44-RNAi. Thus, the two colocalized peptides appear to display similar systemic actions, but differ with respect to feeding and water retention. When knocking down LK in all LK neurons, a very similar set of effects was obtained to those when only the ABLKs were targeted, indicating that in the assays that were performed in this study the other two sets of LK neurons (LHLK and SELK) played a minimal role (Zandawala, 2017).

Interestingly, knockdown of DH44 in ABLKs increases resistance to desiccation and decreases feeding, but these effects were not seen when DH44 was diminished in all DH44 neurons. This is consistent with previous work where inactivation or activation of DH44 neurons had no effect on food intake. Perhaps, the effects seen following ABLK manipulations could be compensated by action of the six DH44-expressing MNCs in the brain. Similarly, reduction of LK in ABLKs causes a slight increase in time of recovery from chill coma, but this is not noted after global knockdown of LK. This minor difference could possibly be attributed to the strength of the two GAL4 driver lines used and, thus, the efficiency of LK knockdown in ABLKs (Zandawala, 2017).

This study also demonstrated that DH44 and LK have additive effects on fluid secretion in MTs. It is likely that these two colocalized peptides are released together and act on the MTs where they target different cell types, receptors, signaling systems, and effectors to regulate fluid secretion. The action of these peptides on the MTs may also in part be responsible for the regulation of stress responses seen in the assays, as shown earlier for CAPA peptide and DH44. It is, however, not clear whether the altered food intake and water retention after DH44 and LK knockdown, respectively, are direct actions on target tissues or indirect effects caused by altered water and ion regulation in the fly (Zandawala, 2017).

Not only do the ABLKs produce two diuretic hormones, but they also seem to be under tight neuronal and hormonal control. Receptors for several neurotransmitters and peptides have been identified on these cells in adults: the serotonin receptor 5-HT1B, LK receptor (LkR), and the insulin receptor dInR. Knockdown of the 5-HT1B receptor in ABLK neurons diminished LK expression, increased desiccation resistance, and diminished food intake, but manipulations of dInR expression in these cells generated no changes in physiology in the tests performed. In larvae, all ABLKs colocalize LK and DH44, and several receptors have been detected in addition to 5-HT1B and dInR [29], namely RYamide receptor, SIFamide receptor, and the ecdysis-triggering hormone (ETH) receptor ETHR-A. However, the expression of these receptors on adult ABLKs has so far not been investigated. Interestingly, the functions of ABLKs in larvae, studied so far, seem to be primarily related to regulating muscle activity and ecdysis motor patterns. The 5-HT-1B receptor on ABLKs was shown to modulate locomotor turning behavior, whereas ETH-mediated activation of ETHR-A on ABLKs initiates the pre-ecdysis motor activity. In this context, it is worth noting that during metamorphosis six to eight novel ABLKs differentiate anteriorly in the abdominal ganglia, and these are the ones that display the strongest expression of DH44. In adult flies, the ABLKs are neurosecretory cells with restricted arborizations in the CNS, but widespread axon terminations along the abdominal body wall and in the lateral heart nerves, whereas in larvae the same cells send axons that terminate on segmental abdominal muscles, muscle 8. It is not yet known whether larval ABLKs are involved in the regulation of diuresis and other related physiological functions in vivo, but certainly larval functions in locomotion and ecdysis behavior are specific to that developmental stage. Thus, it seems that there is a developmental switch of function in this set of peptidergic neuroendocrine cells (Zandawala, 2017).

In summary, this study shows that a set of abdominal neuroendocrine cells, ABLKs, co-expressing DH44 and LK, are sufficient for regulation of resistance to desiccation, starvation and ionic stress, as well as modulating feeding and water content in the body. These ABLKs represent a subset of neurons that express DH44 and LK, and the functions of the remaining neurons are yet to be determined (Zandawala, 2017).

Identification of a circadian output circuit for rest:activity rhythms in Drosophila

Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. A screen was conducted for circadian-relevant neurons in the Drosophila brain, and this study reports that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms (Cavanaugh, 2014).

Given its location near the axonal projections of several groups of clock neurons and its function in metabolic, locomotor, and sleep processes, the PI has been proposed as a possible component of the output pathway in Drosophila, but direct evidence supporting a contribution to behavioral or physiological rhythms has been lacking. This study used a combined genetic, anatomical, and molecular approach to unequivocally identify specific subsets of PI cells as comprising part of the circadian output circuit for rest:activity rhythms. Ectopic activation of PI neurons is sufficient to induce behavioral arrythmicity, and similarly, ablation of small subsets of PI neurons results in loss of rest:activity rhythms. This latter result is consistent with previous studies showing that surgical destruction of the PI in both crickets and cockroaches results in loss of locomotor rhythms. It was further shown that manipulations of the PI that result in behavioral arrhythmicity do not affect the underlying molecular clock in s-LNvs, thus demonstrating that the PI exerts its effects downstream of clock neurons (Cavanaugh, 2014).

Importantly, this study has uncovered a segregation of different behavioral and physiological outputs by specific neurons of the PI. Thus, kurs58-GAL4+ PI neurons function to modulate locomotor behavior, whereas insulin-like peptide-producing PI cells, which constitute a nonoverlapping subset, influence metabolic processes. It will be of interest to determine whether Dilp2+ cells are also modulated by the clock, because such a result would suggest that the PI is a common relay for multiple circadian output circuits that couple to unique physiological functions, each subserved by discrete subpopulations of PI neurons. Furthermore, within kurs58-GAL4+ cells, there appear to be at least two subsets of neurons that contribute to rest:activity cycles. Interestingly, ablation of the SIFa-GAL4+ subset results in reduced rhythmicity, accompanied by decreases in sleep, whereas ablation of the DH44VT-GAL4+ subset also results in reduced rhythmicity, but in this case, the effect on sleep, if any, is an increase. Thus, it is possible that these two molecularly distinct populations control behavioral rhythms through opposing effects on locomotion and/or sleep, and thus, that the contribution of a particular subset predominates depending on time of day (Cavanaugh, 2014).

In conjunction with behavioral studies, GRASP analysis was used to trace neuronal connections emanating from the clock network. It was found that s-LNvs, which function as master pacemakers, make limited connections within the clock cell network and do not appear to directly access output cells of the PI. Instead, PI output cells receive time-of-day information through inputs from DN1 clock cells, as demonstrated by the fact that presynaptic components of DN1 cells adjoin dendrites of PI neurons, in the same brain region where GRASP analysis reveals cellular contacts between these two cell groups. Several studies corroborate a function of DN1 neurons downstream of s-LNvs to mediate rest:activity rhythms. Dorsal neurons are responsive to bath application of PDF, and restoration of the PDF receptor selectively in these neurons of pdfr mutant flies is sufficient to rescue multiple aspects of circadian locomotor rhythms. Furthermore, speeding up the molecular clock in s-LNvs causes concomitant acceleration of molecular cycling in several groups of dorsal neurons, including DN1s. These experiments, along with the current study, argue that DN1 neurons serve an important output function within the clock network and likely reside downstream of s-LNvs in the output circuit for rest:activity rhythms. The data are therefore consistent with a very simple circadian output circuit, in which time-of-day information from the clock network, which is generated by master pacemaker cells (s-LNvs and possibly LNds), passes through dorsal clock neurons (including DN1s) before accessing downstream output neurons of the PI, which then integrate these signals to modulate locomotor rhythms. Whether the PI also lies downstream of other groups of dorsal clock neurons, in addition to DN1s, or whether all time-of-day signals received by the PI pass through DN1 cells remains to be determined (Cavanaugh, 2014).

Within the brain, projections from the PI primarily terminate in the dorsal tritocerebrum; however, more diffuse termination patterns throughout the central brain and optic lobes have been observed for SIFa+ PI neurons. The PI also accesses neurohemal organs via the esophageal canal, as well as directly releasing peptides into the hemolymph. Thus, signals released from the PI could either act within neuronal tissue or systemically via release of peptide neurotransmitters and other hormones. The latter possibility is consistent with studies that showed that transplantation of pers brains into the abdomen of per mutant flies rescued locomotor rhythms, demonstrating that release of a secreted factor underlies brain control of rest:activity rhythms in flies. Similarly, abdominal transplantation of PI cells is sufficient to alter sexually dimorphic locomotor patterns, indicating that the PI can modulate locomotor behavior in a neuroendocrine manner (Cavanaugh, 2014).

Through single-cell transcriptome analysis, the CRF-like peptide, DH44, was identified as a candidate molecule through which PI neurons might influence locomotor behavior. Consistent with this possibility, RNAi-mediated knockdown, or genetic antagonism, of DH44 resulted in altered locomotor behavior and weakened rest:activity rhythms. In addition, selective activation or destruction of DH44+ PI neurons also substantially weakened rest:activity rhythms. In flies, DH44 acts as a diuretic hormone, which stimulates fluid secretion from Malpighian tubules through a cyclic AMP (cAMP) pathway. Its role as a stress molecule is less clear, but DH44 receptor has also been localized to corazonin+ cells of the lateral protocerebrum, which may be involved in the stress response of the fly. Notably, manipulations of neuronal excitability in corazonin+ cells alter stress-induced locomotor activity. In mammals, stress hormones, such as glucocorticoids, show diurnal cycles of secretion and serve as entrainment signals for peripheral clocks. Thus, stress hormones may play a conserved role in circadian regulation of behavioral and physiological processes (Cavanaugh, 2014).

Functional differences between two CRF-related diuretic hormone receptors in Drosophila

In Drosophila, two related G-protein-coupled receptors are members of the corticotropin releasing factor (CRF) receptor subfamily. One of these receptors, encoded by CG8422 (Diuretic hormone 44 receptor 1), has been reported to be a functional receptor for a diuretic hormone, Dh44. This study reports that the other CRF receptor subfamily member, encoded by CG12370 (Diuretic hormone 44 receptor 2), is also a receptor for the Dh44 neuropeptide. The lines of evidence to support this identification include increases in cAMP levels due to CG12370 receptor activation and the recruitment of beta-arrestin-GFP to the plasma membrane in response to Dh44 application. These features of the receptors DH44-R2 (encoded by CG12370) and DH44-R1 (encoded by CG8422) were compared, and fundamental differences were found in signaling, association with the arrestins, and peptide sensitivity. The sensitivity of DH44-R2 to the Dh44 peptide is lower than that of DH44-R1. Previous reports on the sensitivity of the tubule to Dh44 are in agreement with the current measurements of DH44-R2 sensitivity. A specific RNAi construct was used to selectively knock-down DH44-R2 expression and this led to heightened sensitivity to osmotic challenges. The functional characterization of this diuretic hormone receptor in Drosophila demonstrates a high degree of conservation of CRF-like signaling (Hector, 2009).

Diuretic hormone 44 receptor in Malpighian tubules of the mosquito Aedes aegypti: evidence for transcriptional regulation paralleling urination

In the mosquito Aedes aegypti, the molecular endocrine mechanisms underlying rapid water elimination upon eclosion and blood feeding are not fully understood. The genome contains a single predicted diuretic hormone 44 (DH44) gene, but two DH44 receptor genes. The identity of the DH44 receptor(s) in the Malpighian tubule is unknown in any mosquito species. VectorBase gene ID AAEL008292 encodes the DH44 receptor (GPRDIH1) most highly expressed in Malpighian tubules. Sequence analysis and transcript localization indicate that AaegGPRDIH1 is the co-orthologue of the Drosophila melanogaster DH44 receptor (CG12370-PA). Time-course quantitative PCR analysis of Malpighian tubule cDNA revealed AaegGPRDIH1 expression changes paralleling periods of excretion. This suggests that target tissue receptor biology is linked to the known periods of release of diuretic hormones from the nervous system pointing to a common up-stream regulatory mechanism (Jagge, 2008).

Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae

Mosquito natriuretic peptide (MNP), an uncharacterised peptide from the yellow fever mosquito, Aedes aegypti, acts via cyclic AMP to stimulate secretion of Na+-rich urine by opening a Na+ conductance in the basolateral membrane of Malpighian tubule principal cells. Corticotropin releasing factor (CRF)-related peptides and calcitonin (CT)-like diuretic peptides use cyclic AMP as a second messenger and were therefore considered likely candidates for MNP. BLAST searches of the genome of the malaria mosquito Anopheles gambiae, gave sequences for the CRF-related peptide Anoga-DH44 and the CT-like peptide Anoga-DH31, which were synthesised and tested for effects on Malpighian tubules from An. gambiae and Ae. aegypti, together with 8-bromo-cyclic AMP. The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent. Anoga-DH44 and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44 initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31. Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+, whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport. It is concluded that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide (Coast, 2005).

A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling

The Drosophila orphan G protein-coupled receptor encoded by CG17415 is related to members of the calcitonin receptor-like receptor (CLR) family. In mammals, signaling from CLR receptors depend on accessory proteins, namely the receptor activity modifying proteins (RAMPs) and receptor component protein (RCP). The possibility that this Drosophila CLR might also require accessory proteins for proper function was tested; co-expression of the mammalian or Drosophila RCP or mammalian RAMPs permitted neuropeptide diuretic hormone 31 (DH31) signaling from the CG17415 receptor. RAMP subtype expression did not alter the pharmacological profile of CG17415 activation. CG17415 antibodies revealed expression within the principal cells of Malpighian tubules, further implicating DH31 as a ligand for this receptor. Immunostaining in the brain revealed an unexpected convergence of two distinct DH signaling pathways. In both the larval and adult brain, most DH31 receptor-expressing neurons produce the neuropeptide corazonin, and also express the CRFR-related receptor CG8422, which is a receptor for the neuropeptide diuretic hormone 44 (DH44). There is extensive convergence of CRF and CGRP signaling within vertebrates and a striking parallel in Drosophila involving DH44 (CRF) and DH31 (CGRP) is reported. Therefore, it appears that both the molecular details as well as the functional organization of CGRP signaling have been conserved (Johnson, 2005).


Search PubMed for articles about Drosophila Dh44

Cavanaugh, D. J., Geratowski, J. D., Wooltorton, J. R., Spaethling, J. M., Hector, C. E., Zheng, X., Johnson, E. C., Eberwine, J. H. and Sehgal, A. (2014). Identification of a circadian output circuit for rest:activity rhythms in Drosophila. Cell 157: 689-701. PubMed ID: 24766812

Coast, G. M., Garside, C. S., Webster, S. G., Schegg, K. M. and Schooley, D. A. (2005). Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles). J Exp Biol 208: 3281-3291. PubMed ID: 16109890

Dus, M., Lai, J. S., Gunapala, K. M., Min, S., Tayler, T. D., Hergarden, A. C., Geraud, E., Joseph, C. M. and Suh, G. S. (2015). Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila. Neuron. PubMed ID: 26074004

Hector, C. E., Bretz, C. A., Zhao, Y. and Johnson, E. C. (2009). Functional differences between two CRF-related diuretic hormone receptors in Drosophila. J Exp Biol 212: 3142-3147. PubMed ID: 19749107

Jagge, C. L. and Pietrantonio, P. V. (2008). Diuretic hormone 44 receptor in Malpighian tubules of the mosquito Aedes aegypti: evidence for transcriptional regulation paralleling urination. Insect Mol Biol 17: 413-426. PubMed ID: 18651923

Johnson, E. C., Shafer, O. T., Trigg, J. S., Park, J., Schooley, D. A., Dow, J. A. and Taghert, P. H. (2005). A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J Exp Biol 208: 1239-1246. PubMed ID: 15781884

Lee, K.M., Daubnerová, I., Isaac, R.E., Zhang, C., Choi, S., Chung, J. and Kim, Y.J. (2015). A neuronal pathway that controls sperm ejection and storage in female Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25702579

Lovejoy, D. A. and Jahan, S. (2006). Phylogeny of the corticotropin-releasing factor family of peptides in the metazoa. Gen Comp Endocrinol 146: 1-8. PubMed ID: 16472809

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

Zandawala, M., Marley, R., Davies, S. A. and Nassel, D. R. (2017). Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila. Cell Mol Life Sci 75(6):1099-1115. PubMed ID: 29043393

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date revised: 10 April 2017

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