InteractiveFly: GeneBrief

Diuretic hormone 31: Biological Overview | References

BIOLOGICAL OVERVIEW

Body temperature exhibits rhythmic fluctuations over a 24 h period and decreases during the night, which is associated with sleep initiation. However, the underlying mechanism of this temperature decrease is largely unknown. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their body temperature rhythm. This study demonstrates that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, it was previously shown that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, it is proposed that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain (Goda, 2016).

Body temperature rhythm (BTR) is fundamental for maintaining homeostasis, such as in generating metabolic energy and sleep. BTR is one of the most robust circadian outputs and can affect the peripheral clocks of mammals. The rhythmic patterns of BTR and locomotor activity rhythms are analogous. For instance, in diurnal mammals, both body temperature and locomotor activity increase during the daytime and decrease at night. Nonetheless, BTR and locomotor activity rhythms are regulated by different subsets of subparaventricular zone (SPZ) neurons, suggesting that these rhythms are controlled independently (Goda, 2016).

Mammals are not the only examples of this phenomenon. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko, 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their BTR. In Drosophila, TPR and locomotor activity rhythms are regulated by different subsets of clock neurons (Kaneko, 2012). There are ~150 central pacemaker cells in the fly brain, which are functionally homologous to mammalian suprachiasmatic nucleus (SCN) neurons. These pacemaker cells are approximately divided into lateral neurons [LNs; small ventral LNs (s-LNvs), large ventral LNs (l-LNvs), and dorsal lateral neurons (LNds)] and dorsal neurons (DNs; DN1, DN2, and DN3) based on their location and size. The clocks in DN2s are critical for regulating TPR during the daytime (Kaneko, 2012), but not for regulating locomotor activity rhythms (Kaneko, 2012; Goda, 2016 and references therein).

Neuropeptides and their receptors have important roles in synchronizing circadian clocks. A class II G-protein-coupled receptor, pigment-dispersing factor receptor (PDFR), and its ligand (PDF) play important roles in synchronizing circadian clocks and are required for robust circadian locomotor activity in Drosophila. Notably, PDF and PDFR function in a similar manner to vasoactive intestinal peptide (VIP) and its receptor VPAC2 in mammals, both of which play important roles in the ability of clock neurons to regulate the rhythmicity and synchrony of both locomotor activity rhythms and BTRs (Goda, 2016).

Recent reports have suggested that, in addition to PDF, diuretic hormone 31 (DH31) also activates PDFR based on in vitro experiments (Mertins, 2005) and a study that used brain imaging with bath-applied DH31 (Shafer, 2008). Moreover, it has been shown that DH31 is expressed in the posterior dorsal neurons 1 (DN1ps) and that it modulates sleep as a wake-promoting signal before dawn but does not affect locomotor activity rhythms in Drosophila (Kunst, 2014). DH31 is a functional homolog of mammalian calcitonin gene-related peptide (CGRP), which mediates thermosensation and thermoregulation. However, it is unknown whether CGRP is involved in the regulation of BTR in mammals (Goda, 2016).

This study demonstrates that DH31 and PDFR play important roles for TPR at night-onset. DN2s are the main clock cells for TPR, and the data suggest that DH31 binding to PDFR in DN2s regulates temperature preference decreases at night-onset, which is the first in vivo evidence that DH31 could function as a ligand of PDFR. Therefore, it is proposed that circadian locomotor activity and night-onset TPR are regulated by different neuropeptides that use the same receptor expressed in different clock cells (Goda, 2016).

Both Dh31 and Pdfr mutants exhibited abnormal night-onset TPR, and t-DH31 and PDFR expression in DN2s are sufficient to control night-onset TPR, suggesting that DH31 could be a ligand of PDFR in DN2s. Unexpectedly, PDF, an important neuropeptide for locomotor activity, is not required for night-onset TPR. It suggests that PDF and DH31 appear to act on different subsets of PDFR-expressing clock cells to regulate locomotor activity rhythms and night-onset TPR, respectively. Together, the data suggest that locomotor activity and night-onset TPR are regulated through PDFR by different subsets of clock neurons using different neuropeptides. Nonetheless, because all Dh31, Pdf, and Pdfr mutants still maintain normal daytime TPR, the underlying molecular mechanisms of daytime TPR are still obscure (Goda, 2016).

In humans, body temperature dramatically decreases at night, which is associated with sleep initiation. Although it suggests a relationship between BTR and sleep-wake cycles, the underlying mechanisms are largely unclear. A recent study suggested that DH31 mediates sleep-wake cycles and that DH31 secretion functions as a wake-promoting signal before dawn (Kunst, 2014). Given that it was found that DH31 is required for night-onset TPR, DH31 could regulate both sleep-wake cycles and TPR with different timing (i.e., at night-onset for TPR and before dawn for sleep) (Goda, 2016).

While t-DH31 expression in DN2s rescues the Dh31#51 phenotype, t-PDF expression in DN2s slightly rescues in comparison with UAS controls. This shows that t-DH31 in DN2s rescues more efficiently than t-PDF in DN2s. However, DH31 stimulates PDFR less efficiently than PDF in vitro (Mertens, 2005). This suggests that either DH31 may more efficiently bind PDFR in DN2s than PDF or that DN2s may express another receptor of DH31 that also regulates night-onset TPR. Notably, although the DH31 receptor (DH31R) is a known receptor for DH31 in vitro, this study observed that DH31R is not expressed in DN2s and that the Dh31r mutant exhibited normal night-onset TPR. Therefore, it would be interesting to further explore whether additional receptors for DH31 in DN2s are involved in night-onset TPR. It has been previously shown that flies in the light prefer a 1°C higher temperature than in the dark (light-dependent temperature preference [LDTP]), thus demonstrating that light influences temperature preference behavior. Importantly, while LDTP is only affected by light, night-onset TPR is affected by both light and time (ZT10-ZT12 and ZT13-ZT15). Therefore, they are not controlled by the same pathway. In fact, PDFR expression using Clk4.5F-Gal4 rescues the abnormal LDTP phenotype of Pdfr5304 mutants, but it did not rescue night-onset TPR (Goda, 2016).

DH31 is a functional homolog of mammalian CGRP. Importantly, CGRP is related to many physiological functions, such as temperature sensation, migraines, and chronic pain in mammals. CGRP has recently attracted attention for its role in the treatment of migraine attacks, which are associated with body-temperature fluctuations. Additionally, CGRP is expressed in the SCN, but its function in circadian rhythms is not entirely clear. Thus, the findings raise the possibility that CGRP may be involved in BTR regulation and may mediate the connection between the circadian clock and other physiological functions. Moreover, this research will not only expand current understanding of neuropeptidergic regulation, but may also ultimately facilitate a discovery of the fundamental mechanisms of BTR (Goda, 2016).

A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut

The intestine is involved in digestion and absorption, as well as the regulation of metabolism upon sensation of the internal intestinal environment. Enteroendocrine cells are thought to mediate these internal intestinal chemosensory functions. Using the CaLexA (calcium-dependent nuclear import of LexA) method, this study examined the enteroendocrine cell populations that are activated when flies are subjected to various dietary conditions such as starvation, sugar, high fat, protein, or pathogen exposure. A specific subpopulation of enteroendocrine cells in the posterior midgut that express Dh31 and tachykinin was found to be activated by the presence of proteins and amino acids (Park, 2016).

To study the chemosensory functions of enteroendocrine cells in the Drosophila midgut, a method was needed to specifically label as many enteroendocrine cells as possible. In an independent study, Drosophila regulatory peptide genes were sought that are expressed in the enteroendocrine cells. The sum of expression of three regulatory peptide-GAL4 drivers (AstC-, Npf-, and Dh31-GAL4; for convenience, hereafter these three drivers together will be called EE-GAL4) were found to be expressed in about 80% of enteroendocrine cells in the midgut (Park, 2016).

To visualize in vivo changes in cytoplasmic calcium ion concentration, the CaLexA (calcium-dependent nuclear import of LexA) system, which uses a calcium ion-sensitive synthetic transcription factor, was used. When calcium levels rise, modified nuclear factor of activated T cells (NFAT) is imported into the nucleus to transcriptionally activate GFP reporter expression. This CaLexA system has been successfully used to visualize neuronal activation and calcium ion changes in fat body tissue (Park, 2016).

First, whether the CaLexA system could be used to monitor enteroendocrine cell activation, was tested. For this purpose, modified NFAT was expressed in enteroendocrine cells using EE-GAL4. GFP expression in the midgut was monitored after feeding adult flies with 200 mm sucrose for 5 days after eclosion. Sucrose was provided as a minimal nutrient source. Strong GFP expression was observed only in enteroendocrine cells in the middle midgut, while most enteroendocrine cells in the anterior and posterior midgut did not express GFP. Next, whether various dietary conditions could activate the enteroendocrine cells was tested, as well as whether stimuli or enteroendocrine cell specificity exists. Enteroendocrine cell activation in the middle midgut was observed for every tested condition including starvation, indicating that the middle midgut enteroendocrine cells are constitutively activated. The middle midgut is an acidic region, and acid secretion from the enteroendocrine cells likely constitutively occurs to maintain such an environment (Park, 2016).

To quantitate enteroendocrine cell activation using the CaLexA system, the numbers of GFP-expressing cells was counted. GFP-expressing cells in the caudal half of the posterior midgut were counted, since enteroendocrine cell activation upon exposure to various dietary conditions was concentrated to this region. Only GFP-expressing cells costaining with Prospero were counted, excluding autofluorescent signals from food particles. These nonspecific signals can be observed as white dots that do not costain with Prospero, as seen for example in starvation or 50 mm NaCl conditions. When flies were provided with a single sugar diet composed of only sucrose, 14 ± 1.5 SEM GFP-expressing cells were observed in the caudal half of the posterior midgut. This can be considered a baseline for all of the conditions tested, with the exception of starvation, normal fly food, and high fat diet, since 200 mm sucrose was provided in all but these three conditions to induce a quantifiable level of feeding even in adverse conditions. A small number of posterior midgut enteroendocrine cells were activated when cornmeal-based fly food, commonly used in the lab, was provided. Significant activation was not observed when flies were exposed to conditions such as a diet of normal food with high fat composition, starvation, or a diet of 200 mm sucrose with the addition of 50 mm NaCl. In contrast, many enteroendocrine cells were activated in the posterior midgut upon oral infection with Erwinia carotovora carotovora 15 (Ecc15), which causes a gut immune respons. Enteroendocrine cell activation was observed at a similar level when flies were fed Pseudomonas aeruginosa, indicating that the enterondocrine cell response is not specific to a particular bacterial species. Enteroendocrine cell activation was also observed upon feeding on heat-inactivated Ecc15 or P. aeruginosa, at slightly decreased levels compared to untreated bacteria, but still higher than the 200 mm sucrose control flies. In contrast, enteroendocrine cells were not activated upon flies being fed uracil, which causes a gut immune response through acting as a DUOX-activating ligand in the Drosophila gut epithelia. These results indicated that enteroendocrine cell activation is not due to the detection of pathogenicity. Supporting this conclusion, ingestion of nonpathogenic E. coli or yeast also caused enteroendocrine cell activation. On the basis of these results, it is hypothesized that the enteroendocrine cells were being activated by the presence of protein. Enteroendocrine cell activation was observed in a dose-dependent manner when flies were fed various concentrations of casein peptone, providing evidence that protein cues were activating the enteroendocrine cells. Next, the twenty amino acids were all individually tested for enteroendocrine cell activation. The amino acids caused enteroendocrine cell activation, indicating that enteroendocrine cells are activated upon detection of most, if not all, amino acids. Aspartic acid and glutamic acid, which contain negatively charged side chains, caused a relatively low number of activated cells, but this is likely due to the lower concentration used due to solubility issues. The number of activated cells also increased in a dose-dependent manner, suggesting that these amino acids act as cues to activate enteroendocrine cells. For similar reasons, poorly water-soluble tyrosine also cannot be excluded as a potential activation cue for enteroendocrine cells (Park, 2016).

Enteroendocrine cells in the posterior midgut are activated by protein and amino acids. GFP expression pattern induced by the CaLexA system in the caudal half of the posterior midgut upon exposure to the indicated dietary conditions (Park, 2016).

Enteroendocrine cell activation by microorganisms, casein peptone, and amino acids was concentrated in enteroendocrine cells of the posterior midgut, in particular the caudal half. Enteroendocrine cells in this region can be largely divided into two populations, with one population expressing the regulatory peptides AstA and AstC, and the other expressing Dh31 and tachykinin. To examine which population of enteroendocrine cells in the caudal half of the posterior midgut are activated by microorganisms, casein peptone, and amino acids, the enteroendocrine cells were costained with AstA or Dh31 antisera. Although EE-GAL4 is expressed in both populations of enteroendocrine cells in the caudal half of the posterior midgut, most of the activated enteroendocrine cells belonged to the Dh31-expressing cell population. When Ecc15 was provided, an average of 68 cells label with Dh31 antiserum in the corresponding region, and 54 out of 61 cells that express GFP through the CaLexA system co-stain with Dh31 antiserum. Under the same conditions, an average of 96 cells are labeled by AstA antiserum, and only two of 64 cells expressing GFP through the CaLexA system co-stain with AstA antiserum . In conclusion, amino acids in proteins activate a specific subset of Dh31- and tachykinin-expressing enteroendocrine cells in the posterior midgut through an as yet unknown mechanism. Since the activation of these cells appears to be unrelated to pathogenicity of the protein source, it seems unlikely that these enteroendocrine cells are directly involved in eliciting an immune response to pathogens. The role of this enteroendocrine cell subgroup thus appears to mainly be detection of a potential nutrient source through its protein content. It is unclear whether activation of these cells by amino acids influences the production and/or secretion of Dh31 or other regulatory peptides. Weaker Dh31 antibody staining was not observed in GFP-expressing cells, which would be expected if Dh31 secretion was enhanced in the activated cells. It is formally possible that Dh31 secretion is enhanced in activated cells, and production is increased to compensate for the increased secretion, but the data are insufficient to provide support for either scenario. Tachykinin production is enhanced in the midgut upon nutrient deprivation, resulting in repression of lipogenesis in enterocytes. However, in this study, the enteroendocrine cells activated by proteins and amino acids were not activated upon starvation, and sucrose was coprovided as a minimal nutrient in all dietary conditions providing proteins and amino acids, with the sucrose-only basal level acting as a baseline for activation. This indicates that proteins and amino acids, and not other cues such as carbohydrate deprivation, act as specific cues to activate a particular subset of enteroendocrine cells. The molecular identity of potential protein or amino acid receptors in the enteroendocrine cells is as yet unknown. In mammals, several amino acids including L-glutamine have been shown to stimulate GLP1 secretion in vitro. CaSR, a primarily Gq-coupled calcium-sensing receptor, is expressed in the enteroendocrine cells, and CaSR activation has been associated with amino acid-stimulated gut hormone secretion. The umami taste receptor dimer T1R1/T1R3 and GPRC6A are also additional candidates for mediating GLP1 release in response to amino acids. This work provides an in vivo enteroendocrine system to investigate such possible amino acid receptors (Park, 2016).

This study has shown that the CaLexA system can be used to monitor the activation of Drosophila midgut enteroendocrine cells upon exposure to specific stimuli. Activation of enteroendocrine cells upon exposure to sugar, fat, and bitter compounds was not observed using this method. It was found that proteins and most amino acids are capable of activating enteroendocrine cells in the posterior midgut. These activated cells are limited to a subpopulation of enteroendocrine cells that secrete specific regulatory peptides including Dh31 and tachykinin. This study provides an important step in studying the chemosensory functions of enteroendocrine cells (Park, 2016).

Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila

Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive. However, very little is known about the cellular and molecular mechanisms by which the circadian clock regulates sleep. This study describes a novel role for Diuretic hormone 31 (DH31), the fly homolog of the vertebrate neuropeptide calcitonin gene-related peptide, as a circadian wake-promoting signal that awakens the fly in anticipation of dawn. Analysis of loss-of-function and gain-of-function Drosophila mutants demonstrates that DH31 suppresses sleep late at night. DH31 is expressed by a subset of dorsal circadian clock neurons that also express the receptor for the circadian neuropeptide pigment-dispersing factor (PDF). PDF secreted by the ventral pacemaker subset of circadian clock neurons acts on PDF receptors in the DH31-expressing dorsal clock neurons to increase DH31 secretion before dawn. Activation of PDF receptors in DH31-positive DN1 specifically affects sleep and has no effect on circadian rhythms, thus constituting a dedicated locus for circadian regulation of sleep. This study has identified a novel signaling molecule (DH31) as part of a neuropeptide relay mechanism for circadian control of sleep. The results indicate that outputs of the clock controlling sleep and locomotor rhythms are mediated via distinct neuronal pathways (Kunst, 2014).

Vertebrate Calcitonin gene-related peptide (CGRP) has been implicated in controlling anxietyand stress response. While not previously addressed experimentally, the intimate relationship between stress, anxiety, and sleep suggests that CGRP might regulate sleep. Potentially relevant to this possibility is the recent observation that acute activation of CGRP signaling in zebrafish larvae increases spontaneous locomotor activity and decreases quiescence. This analysis of gain-of-function and loss-of-function mutant flies establishes that DH31, the Drosophila homolog of CGRP, is a negative regulator of sleep maintenance that awakens the animal in anticipation of dawn. This finding motivates investigation of a potential role for CGRP in the regulation of vertebrate sleep (Kunst, 2014).

Using powerful tools for cell-specific neuronal manipulation, this study identified a highly restricted subset of DN1 circadian clock neurons that secrete DH31 late at night to awaken the fly in anticipation of dawn. Neuropeptides secreted from the circadian pacemaker in the mammalian SCN, such as prokinecticin 2 and cardiotrophin-like cytokine, are important clock outputs, but their cellular targets and molecular mechanisms remain unknown. In flies, PDF-expressing sLNv pacemaker neurons are known to be upstream of DN1s, sLNvs are most active around dawn, and PDF secretion from LNvs suppresses sleep at night. However, how PDF signals propagate out of the circadian clock network to regulate sleep remains unknown (Kunst, 2014).

This study shows that PDF secreted by the sLNv pacemaker neurons activates PDFR in the DH31-expressing DN1s to increase neuronal activity and DH31 secretion late at night, thereby awakening the fly in anticipation of dawn. This is consistent with an earlier report demonstrating that flies lacking PDF or PDFR sleep more late at night. However, unlike DH31, PDF also promotes wake during the day. This suggests that PDF regulation of daytime sleep is mediated by neurons other than the DH31-expressing DN1s. PDFR is expressed by circadian clock neurons in addition to DN1s, and PDF signaling to these other clock neurons could be responsible for the wake-promoting effect of PDF during the day (Kunst, 2014).

While PDF plays a key role in circadian timekeeping, neither constitutive activation of PDFR in the DH31-expressing DN1s nor manipulation of their secretion of DH31 affects free-running circadian rhythms. This establishes the PDF-to-DH31 neuropeptide relay as a novel sleep-specific output of the circadian pacemaker network, independent from and parallel to the outputs that drive circadian rhythms themselves. Since the basic cellular and molecular organization of vertebrate and insect circadian networks is conserved, this motivates the search for similar mechanisms in the SCN. Future studies are required to identify the neuronal targets of sleep-regulating DH31 signals and the cellular and molecular mechanisms by which DH31-R1 activation in these targets induces wake (Kunst, 2014).

Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity

Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in Drosophila locomotor activity. This study demonstrates that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. It was determined that Dh31 loss-of-function mutants (Dh31^#51) showed normal rhythmicity, whereas Dh31(^#51);Pdf⁰¹ double mutants exhibited a severe arrhythmic phenotype compared to Pdf-null mutants (Pdf⁰¹). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of Dh31(^#51);Pdf⁰¹double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in Dh31(^#51);Pdf⁰¹ mutants were similar to those in Pdf⁰¹ mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in Dh31 receptor (Dh31r) expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the Pdf receptor (Pdfr) mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, this study identifies a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps (Goda, 2019).

This study has demonstrated a novel function of DH31 in regulating Drosophila locomotor activity rhythms. Dh31^#51 mutants maintained a robust free-running rhythm, whereas Dh31^#51;Pdf⁰¹ double-mutant flies exhibited a severe disruption of their free-running rhythm compared to Pdf⁰¹ mutants. These findings suggest that Dh31^#51 mutants maintain a robust free-running rhythm because the primary factor, PDF, can sustain a strong rhythm. ~40% of Pdf⁰¹ single-mutant flies exhibited a preserved rhythmic state, which is because DH31 can partially support free-running rhythmicity. Thus, the severe disruptions of free-running rhythm in Pdf⁰¹ and Dh31^#51 double-mutant flies is likely caused by the loss of both pathways (Goda, 2019).

PDF is secreted from the main circadian neurons, LNvs, and acts on other clock cells through PDFR to synchronize and maintain robust molecular rhythms. PDF expression from LNvs in Dh31^#51;Pdf⁰¹ mutants restored rhythmicity, in contrast to tethered-PDF (t-PDF) expression in LNvs, indicating that an autoreceptor of PDF signals in LNvs is not sufficient to maintain rhythmicity. Instead, t-PDF expression in DN1ps restored rhythmicity, suggesting that PDF signaling in DN1ps is sufficient to maintain robust free-running rhythmicity. Recently, the responsiveness to PDF was shown to be strongly altered for 24 h via RalA GTPase in sLNvs28. Therefore, it is expected that the continuous activation of PDFR by t-PDF generates rhythmic downstream signaling in PDFR-expressing neurons (Goda, 2019).

Molecular oscillations in DN1s were strongly dampened in Pdf⁰¹ mutants compared with WT flies. These data are consistent with previous studies in which the molecular oscillations of PER in Pdf⁰¹ mutants held under DD conditions were dampened in DN1s10 and the genetic manipulation of the circadian clocks in PDF-positive cells altered the molecular rhythms in DN1ps. Furthermore, Pdfr expression in DN1ps has been reported to prevent the arrhythmic phenotype in Pdfr⁵³⁰⁴ mutants. These findings support the idea that PDF is secreted from LNvs and acts on DN1ps to regulate free-running rhythmicity (Goda, 2019).

Furthermore, it was shown that t-DH31 expression in DN1ps rescued the Pdf⁰¹ and Dh31^#51 double-mutant phenotypes, which suggests that DH31 acts on DN1ps to regulate rhythmicity. Although it has been suggested that DH31 release might increase at dawn and that DH31-mRNA expression levels oscillate for 24 h, how t-DH31 expression causes rhythmic behavioral output remains unclear. Because DH31 can modestly activate PDFR in vitro, it cannot be excluded that t-DH31 overexpression might simply activate PDFR in DN1ps instead of the intrinsic PDF signals, thereby restoring locomotor activity rhythms in the flies. However, the rhythmicity of Dh31^#51;Pdf⁰¹ mutants overexpressing t-DH31 in tim-Gal4-expressing neurons or R18H11-Gal4-expressing DN1ps only reached levels similar to that of the Pdf⁰¹ single-mutant flies. Therefore, DH31 likely acts on DN1ps separately from the PDF pathway (Goda, 2019).

Although it has been shown that DH31 is expressed in a subset of DN1ps, DH31 expression using R18H11-Gal4 did not rescue the Pdf⁰¹ and Dh31^#51 double-mutant phenotypes, suggesting that DH31 expression in R18H11-Gal4-expressing neurons is insufficient to maintain rhythmicity. Instead, DH31 is expressed in DN1as and DH31 expression in tim-Gal4-expressing neurons rescued the phenotype, which suggests that DH31 expression in clock neurons maintains rhythmicity. That said, given that DH31 is expressed in nonclock neurons and that tim-Gal4 is expressed in nonclock cells, it cannot be excluded that DH31 expression in nonclock neurons might play a role in rescuing the severe phenotype of Dh31^#51;Pdf⁰¹ mutants. Alternatively, although DH31 expression in LNvs was not detectable via anti-DH31 antibody staining, a recent RNA-seq analysis detected Dh31 gene expression in both LNvs and DN1s. Therefore, DH31 expression from LNvs may potentially act on DN1s to support locomotor activity rhythms (Goda, 2019).

In summary, it is proposed that PDF and DH31 regulate free-running rhythms in a hierarchical fashion in DN1ps. As t-DH31 or t-PDF expression in DN1ps resulted in a similar level of rhythmicity as that observed in flies expressing t-DH31 or t-PDF, respectively, in tim-Gal4-expressing neurons, DN1ps are at least one of the important clock cells that regulate free-running rhythmicity (Goda, 2019).

Given that Dh31^#51;Pdf⁰¹ mutants exhibited severe arrhythmicity in free-running rhythm, it is speculated that the severe arrhythmic phenotype might be a result of abnormal molecular oscillations. However, the molecular oscillations of Dh31^#51;Pdf⁰¹ mutants were similar to those of Pdf⁰¹ mutants. Therefore, the molecular mechanisms by which DH31 regulates free-running rhythms still remain unclear. Importantly, the peak of VRI expression in LNds in Dh31^#51 was at ZT 19, which was delayed compared with those of WT flies and the other mutants. The data suggested that DH31 is involved in the regulation of molecular oscillations in LNds. Because LNds are the evening pacemaker, the delayed VRI oscillations in LNds might be associated with the longer period of free-running rhythm in Dh31^#51 (Goda, 2019).

Recently, the intracellular calcium rhythms in each clock cell were reported to be nonsynchronous and associated with morning and evening peaks in locomotor activity. DH31 signaling may possibly contribute to the downstream output that controls molecular rhythms in pacemaker processes, such as intracellular calcium rhythms. Given that PDF from sLNvs regulates strong molecular rhythms in DN1ps and generates robust free-running rhythms under constant conditions, DH31 may help maintain vigorous output signals downstream of the molecular clocks in DN1ps (Goda, 2019).

Recently work has shown that both Dh31r^1/Df mutants and flies undergoing Dh31r knockdown in their neurons showed normal rhythmicity in the locomotor activity rhythm25. In contrast to Dh31^#51;Pdf⁰¹ double mutants, Pdfr5304;Dh31r^1/Df double mutants did not enhance the arrhythmicity observed in Pdfr single mutants, which suggests that Dh31r does not complement PDFR function; thus, Dh31r does not function as a receptor for DH31 in this context. Given that Dh31r^1/Df flies showed a strong abnormality in the TPR phenotype25, it is more likely that Dh31r does not play an important role in locomotor activity rhythms. However, Dh31r^1/Df4 mutants are not null25, and it cannot be excluded that a small amount of residual Dh31r might drive robust locomotor activity rhythms with the PDF pathway (Goda, 2019).

Which receptors might function with DH31 to regulate free-running rhythmicity? Given that DH31 can activate PDFR in vitro, bath applications of DH31 can activate LNvs via PDFR19 and DH31 can function as a ligand of PDFR in TPR at the onset of night, PDFR may function as a receptors for both DH31 and PDF in the regulation of free-running rhythmicity. However, because the arrhythmicity of Pdfr5304 mutants was not as severe as that of Dh31^#51;Pdf⁰¹ mutants, PDFR does not appear to act as a receptor for DH31 in this context (Goda, 2019).

Both Dh31r and PDFR are class II G-protein coupled receptors (GPCRs), which also include Hector and Diuretic hormone 44 receptors 1 and 2 (DH44R1 and DH44R2, respectively). Interestingly, the DH44R1 and DH44R2 ligand DH44 has been implicated in circadian output circuits. Therefore, although there is no evidence from in vitro or in vivo experiments, these receptors might nevertheless function as receptors for DH31 to regulate free-running rhythmicity (Goda, 2019).

Orchestration of neuropeptides regulates locomotor activity rhythms in species ranging from flies to mammals The orchestration of neuropeptides is critical for regulating circadian clock functions in species that range from flies to mammals. In mammals, several neuropeptides, including vasoactive intestinal polypeptide (VIP), arginine vasopressin (AVP) and neuromedin S (NMS), are expressed in the SCN, which is the center for circadian clock control. The hierarchy of neuropeptide signaling contributes to circadian function in the SCN. Several recent studies in Drosophila have identified the neuropeptides, including ion transport peptide (ITP), neuropeptide F (NPF), allatostatin A, short neuropeptide F, leucokinin and DH44, that regulate locomotor activity and sleep. However, given that DH31 complements the function of PDF in regulating free-running rhythmicity in the same clock cells, DH31 not only serves as one of the neuropeptides that regulates circadian rhythms but also might selectively influence PDF function in the regulation of free-running rhythms. Thus, these findings shed new light on the next steps required to improve understanding of the core neuropeptide regulatory mechanisms involved in the circadian rhythm (Goda, 2019).

Effects of diuretic hormone 31, drosokinin, and allatostatin A on transepithelial K(+) transport and contraction frequency in the midgut and hindgut of larval Drosophila melanogaster

Recent studies have identified paracrine and endocrine cells in the midgut of larval Drosophila melanogaster as well as midgut and hindgut receptors for multiple neuropeptides implicated in the control of fluid and ion balance. Although the effects of diuretic factors on fluid secretion by isolated Malpighian tubules of D. melanogaster have been examined extensively, relatively little is known about the effects of such factors on gut peristalsis or ion transport across the gut. The effects were measured of diuretic hormone 31 (DH31), drosokinin and allatostatin A (AST-A) on both K(+) transport and muscle contraction frequency in the isolated gut of larval D. melanogaster. K(+) absorption across the gut was measured using K(+) -selective microelectrodes and the scanning ion-selective electrode technique. Allatostatin A (AST-A; 1 mμM) increased K(+) absorption across the anterior midgut but reduced K(+) absorption across the copper cells and large flat cells of the middle midgut. AST-A strongly inhibited gut contractions in the anterior midgut but had no effect on contractions of the pyloric sphincter induced by proctolin. DH31 (1 mμM) increased the contraction frequency in the anterior midgut, but had no effect on K(+) flux across the anterior, middle, or posterior midgut or across the ileum. Drosokinin (1 μM) did not affect either contraction frequency or K(+) flux across any of the gut regions examined. Possible functions of AST-A, DH31, and drosokinin in regulating midgut physiology are discussed (Vanderveken, 2014).

Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging

The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of approximately 150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. This question was addressed by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. A subset of the PDF-expressing neurons was found to respond to PDF with long-lasting cAMP increases, and such responses were confirmed require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus, the network of approximately 150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with subcellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network (Shafer, 2008).

PDF receptor signaling in Drosophila contributes to both circadian and geotactic behaviors

The neuropeptide Pigment-Dispersing Factor (PDF) is a principle transmitter regulating circadian locomotor rhythms in Drosophila. A Class II (secretin-related) G protein-coupled receptor (GPCR) has been identified that is specifically responsive to PDF, to PACAP and a to Drosophila ortholog of calcitonin called DH31. In response to PDF, the PDF receptor (PDFR) elevates cAMP levels when expressed in HEK293 cells. As predicted by in vivo studies, cotransfection of Neurofibromatosis Factor 1 significantly improves coupling of PDFR to adenylate cyclase. pdfr mutant flies display increased circadian arrhythmicity, and also display altered geotaxis that is epistatic to that of pdf mutants. PDFR immunosignals are expressed by diverse neurons, but only by a small subset of circadian pacemakers. These data establish the first synapse within the Drosophila circadian neural circuit and underscore the importance of Class II peptide GPCR signaling in circadian neural systems (Mertens, 2005).

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

The Drosophila orphan G protein-coupled receptor encoded by CG17415 (Diuretic hormone 31 Receptor) 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).

The CLR encoded by Drosophila CG17415 is activated by the neuropeptide DH₃₁. The evidence includes functional responses derived from both signaling and desensitization assays. The identification of CG17415 as a DH31-R1 was further supported by the demonstration that it is expressed in principal cells of the Malpighian tubules, which were previously shown to be sensitive to the DH₃₁ peptide. Notably, it was found that signaling by DH31-R1 in HEK-293 is dependent upon co-expression of additional subunits, specifically RCP. This finding argues that RCP association is a widespread feature of CLR signaling across large phylogenetic distances. Consonant with this proposition, the genomes of Apis and Anopheles contain orthologs of the RCP receptor and DH₃₁ peptide . Additionally, a receptor that was recently cloned from the bivalve Crassostrea gigas shows high sequence similarity to calcitonin and CGRP receptors, and to Drosophila DH31-R1 (CG17415). The expression of this receptor was described in various tissues and the authors speculated on its potential role in ionic balance in that mollusc. This finding argues that this fundamental feature of CLR signaling predates the separation of arthropods from chordates (Johnson, 2005).

When expressed in NIH 3T3 cells, CG17415 did respond to DH₃₁ without co-transfection of RCP or RAMPs; this can be attributed to higher endogenous levels of RCP and RAMPs. How CLR accessory proteins such as RCP and RAMPs promote CLR functions remains uncertain; RCP may couple the receptor to the G_s protein, or it may activate adenylate cyclase directly. In mammals, RCP expression largely mirrors that of CLR, whereas RAMP expression typically exceeds that of CLR. Consistent with the expectation that RAMPs have a larger set of functions than does RCP, RAMPs interact with more than the CLR receptor and are known to interact with the VPAC-1, PTH-1 receptor and glucagon receptor (Johnson, 2005).

This study found that RAMP co-expression permitted detection of CG17415 receptor activity without affecting its pharmacological profile. A comparable situation occurs for the neuropeptide intermedin (also referred to as adrenomedullin 2), which is related to CGRP. RAMP co-expression with CLR in HEK cells permitted functional responses, but RAMP subtype did not alter the pharmacological response of the CLR to intermedin (Johnson, 2005).

The current lack of RAMP candidates in the Drosophila genome indicates either their true absence, or that diagnostic structural characteristics of RAMPs have not been conserved. The results show that the Drosophila CLR is able to interact functionally with human RAMPs in HEK cells, and it is therefore considered possible that Drosophila utilizes RAMP-like proteins. Given the promiscuity that many members of Family B receptors demonstrate for different ligands, it is not possible to rule out the possibility of additional peptide ligands for this receptor (which may be dependent upon a Drosophila RAMP), nor can additional DH₃₁ receptors be ruled out. It is noted there are two remaining orphan CLR-related receptors in the Drosophila genome (CG4395 and CG13758) (Johnson, 2005).

Another similarity between Drosophila CG17415 and the mammalian CLR is that they both appear to require the RCP and RAMP accessory subunits for desensitization as well as for signaling. While the demonstration of CG17415 desensitization (indirectly measured by the recruitment of β-arrestin-GFP) may not be indicative of the situation occurring in native cells, it does represent an additional measure of specific receptor activation by the DH₃₁ peptide. The pattern of β -arrestin2 association was observed to be typical for a Family B receptor (Johnson, 2005).

The immunocytochemical demonstration of DH31-R expression in the principal cells of the Malpighian tubules also supports the identification of DH₃₁ as a ligand. A recent study using microarray analysis corroborates this finding; CG17415 transcript levels are enriched 17-fold within the tubule. It was not possible to detect DH44-R1 immunosignals within the principal cells of the tubule, which is consonant with the transcriptional profile of this tissue. A potential second DH₄₄ receptor that could mediate DH₄₄ activation of cAMP on the tubule is encoded by CG12370. It is noted that in functional assays the estimates of EC50 values are larger than values derived from physiological assays on DH₃₁ sensitivity on isolated Malpighian tubules. However, the finding that human RCPs and RAMPs supported uniformly larger amplitude responses (compared to the effect of co-expressed Drosophila RCP) leads to a suggestion that such differences probably derive from issues of expression in a heterologous (e.g. mammalian) cellular context. It is possible that the human accessory proteins are better able to couple with downstream effectors in a human cell line than in the Drosophila accessory protein. Thus, it is argued that the Drosophila RCP probably represents a fundamental component of in vivo DH₃₁ signaling (Johnson, 2005).

The co-expression of DH₃₁ (CG17415) and DH₄₄ (CG8422) receptors within CRZ neurons suggests clear, functional hypotheses. First, it indicates that both the DH₃₁ and DH₄₄ signaling pathways play unexpected roles to facilitate or inhibit CRZ release. Second, the fact that all CRZ neurons express both receptors indicates a close association between CRZ signaling functions and upstream regulation by convergent DH₃₁ and DH₄₄ signaling pathways. Third, the fact that a large fraction of DH receptor-positive neurons are CRZ cells suggests that much of the DH receptor signaling within the Drosophila CNS is dedicated to regulation of CRZ release. Recent work has shown a co-localization of different diuretic peptides in various insects. In Drosophila, DH₄₄ is strictly co-localized with the leucokinin receptor. That observation reinforces the general conclusion that the functional interactions between these diuretic regulatory peptides in the periphery may have counterparts within neural circuits of the CNS. Whether the CRZ-expressing neurons contribute to the neural control of diuresis, or are involved in unrelated physiology, is uncertain. Corazonin is a multi-functional peptide that helps initiate ecdysis; is expressed in clock neurons in Manduca, is correlated with pigmentation state in Locusta and is cardioactive in Periplaneta (Johnson, 2005).

A convergence of CLR (DH₃₁) receptor and CRF (DH₄₄) receptor signaling within functional neural circuits is not unprecedented. These two signaling pathways coincide at several distinct loci within the mammalian CNS and pituitary. For example, in the vestibular cerebellar cortex of mice, CGRP and CRF-like immunostaining innervate non-overlapping domains of Purkinje cell dendrites during development. CRF mediates the CGRP-induced increase in corticosterone release. Likewise, CRF helps mediate the CGRP suppression of pulsatile LH secretion via gonadotropin releasing hormone (GnRH) neurons. It is noted that the receptor for the CRZ neuropeptide is ancestrally related to GnRH receptors. CGRP terminals from pontine/parabrachial nucleus innervate CRF neurons of the amygdyla, and both peptides in this pathway cause increases in autonomic outflow, including increases in heart rate and blood pressure. This detail is also notable since CRZ is a cardioactive factor. Together, these observations are consistent with the hypothesis that the convergence of CLR and CRF-R signaling pathways is a conserved feature in the evolution of neural circuits. Further study of these signaling pathways in Drosophila may therefore contribute to a fundamental understanding of the peptide circuits across phylogeny. (Johnson, 2005).

Search PubMed for articles about Drosophila DH31

Goda, T., Tang, X., Umezaki, Y., Chu, M. L. and Hamada, F. N. (2016). Drosophila DH31 neuropeptide and PDF receptor regulate night-onset temperature preference. J Neurosci 36: 11739-11754. PubMed ID: 27852781

Goda, T., Umezaki, Y., Alwattari, F., Seo, H. W. and Hamada, F. N. (2019). Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity. Sci Rep 9(1): 838. PubMed ID: 30696873

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

Kaneko, H., Head, L. M., Ling, J., Tang, X., Liu, Y., Hardin, P. E., Emery, P. and Hamada, F. N. (2012). Circadian rhythm of temperature preference and its neural control in Drosophila. Curr Biol 22(19): 1851-1857. PubMed ID: 22981774

Kunst, M., Hughes, M. E., Raccuglia, D., Felix, M., Li, M., Barnett, G., Duah, J. and Nitabach, M. N. (2014). Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila. Curr Biol 24: 2652-2664. PubMed ID: 25455031

Mertens, I., Vandingenen, A., Johnson, E. C., Shafer, O. T., Li, W., Trigg, J. S., De Loof, A., Schoofs, L. and Taghert, P. H. (2005). PDF receptor signaling in Drosophila contributes to both circadian and geotactic behaviors. Neuron 48(2): 213-219. PubMed ID: 16242402

Park, J. H., Chen, J., Jang, S., Ahn, T. J., Kang, K., Choi, M. S. and Kwon, J. Y. (2016). A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut. FEBS Lett 590: 493-500. PubMed ID: 26801353

Shafer, O. T., Kim, D. J., Dunbar-Yaffe, R., Nikolaev, V. O., Lohse, M. J. and Taghert, P. H. (2008). Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging. Neuron 58(2): 223-237. PubMed ID: 18439407

Vanderveken, M. and O'Donnell, M. J. (2014). Effects of diuretic hormone 31, drosokinin, and allatostatin A on transepithelial K(+) transport and contraction frequency in the midgut and hindgut of larval Drosophila melanogaster. Arch Insect Biochem Physiol 85(2): 76-93. PubMed ID: 24408875

date revised: 21 June 2024

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