Anti-diuretic hormone ITP signals via a guanylate cyclase receptor to modulate systemic homeostasis in Drosophila

Metabolic and osmotic homeostasis are under strict control in organisms to ensure fitness and survival, as well as promote growth and reproduction. Homeostasis is achieved by regulatory mechanisms that impart plasticity to behaviors such as foraging, feeding, drinking, defecation, and physiological processes, including digestion, energy storage/mobilization, and diuresis. For any given homeostatic system, deviations from the optimal range are monitored by external and internal sensors. These, in turn, signal to central neuronal circuits where information about the sensory stimuli and internal states is integrated. The associated regulatory output pathways commonly utilize neuropeptides or peptide hormones to orchestrate appropriate behavioral and physiological processes (Rajan and Perrimon, 2011; Sternson, 2013a; Jourjine et al., 2016; Lin et al., 2019; Nässel and Zandawala, 2019; Miroschnikow et al., 2020; Benevento et al., 2022; McKim et al., 2024). In mammals, hypothalamic peptidergic neuronal systems, in conjunction with peptide hormones released from the pituitary, are critical regulators of feeding, drinking, metabolic and osmotic homeostasis and reproduction (Sternson et al., 2013b; Saper and Lowell, 2014; Le Tissier et al., 2017; Benevento et al., 2022). Several peptidergic pathways have also been delineated in insects that regulate similar homeostatic functions (Rajan and Perrimon, 2011; Schooley et al., 2012; Schoofs et al., 2017; Lin et al., 2019; Nässel and Zandawala, 2019; Nässel and Zandawala, 2020; Kim et al., 2021; Koyama et al., 2023). Some of these insect pathways originate in the neurosecretory centers of the brain and the ventral nerve cord (VNC), as well as in other endocrine cells located in the intestine (Raabe, 1989; Hartenstein, 2006; Zandawala et al., 2018a; Nässel and Zandawala, 2020; Zandawala et al., 2021; Koyama et al., 2023; McKim et al., 2024). Additionally, peptidergic interneurons distributed across the brain also play important roles in regulation of homeostatic behavior and physiology (Schlegel et al., 2016; Martelli et al., 2017; Lin et al., 2019; Yurgel et al., 2019; Miroschnikow et al., 2020; Nässel and Zandawala, 2020). Importantly, some insect neuropeptides are released by both interneurons and neurosecretory cells (NSC), indicating central and hormonal roles, respectively. One such example is the multifunctional ion transport peptide (ITP).

ITP derived its name from its first determined function in the locust Schistocerca gregaria, where it increases chloride transport across the ileum and acts as an anti-diuretic hormone (Audsley et al., 1992b). Subsequent studies in other insects identified additional roles of ITP, including in reproduction, development, and post-ecdysis behaviors (Begum et al., 2009; Yu et al., 2016b). In Drosophila, ITP influences feeding, drinking, metabolism, and excretion (Gáliková et al., 2018; Gáliková and Klepsatel, 2022). Moreover, it has a localized interneuronal role in the Drosophila circadian clock system (Johard et al., 2009; Hermann-Luibl et al., 2014; Reinhard et al., 2024). The Drosophila ITP gene encodes five transcript variants, which generate three distinct peptide isoforms: one ITP amidated (ITPa) isoform and two ITP-like (ITPL1 and ITPL2) isoforms (Dircksen et al., 2008; Gramates et al., 2022; Figure 1A). ITPa is a C-terminally amidated 73 amino acid neuropeptide, while ITPL1 and ITPL2 are non-amidated and possess an alternate, extended C-terminus. ITPa and ITPL isoforms are also found in other insects, indicating conservation of the ITP splicing pattern (Dai et al., 2007). Moreover, insect ITP is homologous to crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH), which together form a large family of multifunctional neuropeptides (Meredith et al., 1996; Dai et al., 2007; Drexler et al., 2007; Dircksen et al., 2008; Begum et al., 2009; Webster et al., 2012).

Figure 1

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ITPa is produced by a small set of interneurons and NSC in the Drosophila brain and VNC (Dircksen et al., 2008). While the expression of the ITPL peptides has not yet been investigated in Drosophila, studies in other insects indicate partial overlap with ITPa-expressing neurons (Drexler et al., 2007; Klöcklerová et al., 2023). In order to delineate the targets of ITPa and ITPL and determine their modes of action, it is first necessary to identify, functionally characterize, and localize the distribution of their cognate receptors. ITPa and ITPL receptors have been characterized in the silk moth Bombyx mori (Nagai et al., 2014). Surprisingly, Bombyx ITPa and ITPL were found to activate G-protein-coupled receptors (GPCRs) for pyrokinin and tachykinin neuropeptides, respectively (Nagai et al., 2014; Nagai-Okatani et al., 2016). Recently, ITPL2 was also shown to exert anti-diuretic effects via the tachykinin receptor 99D (TkR99D) in a Drosophila tumor model (Xu et al., 2023). Given the lack of structural similarity between ITPa/ITPL, pyrokinin and tachykinin, the mechanisms governing crosstalk between these diverse signaling pathways are still unclear. More importantly, the presence of any additional ITPa receptors in insects is so far unknown.

Here, we address these knowledge gaps by comprehensively characterizing ITP signaling in Drosophila. We used a combination of anatomical mapping and single-cell transcriptome analyses to localize expression of all three ITP isoforms in the nervous system and peripheral tissues. Importantly, we also functionally characterized the membrane-associated receptor guanylate cyclase, Gyc76C, and identified it as a Drosophila ITPa receptor. We show that ITPa-Gyc76C signaling to the fat body and renal tubules influences metabolic and osmotic homeostasis, respectively. Lastly, we identified synaptic and paracrine input and output pathways of ITP-expressing neurons using connectomics and single-cell transcriptomics, thus providing a framework to understand how ITP neurons integrate diverse inputs to orchestrate systemic homeostasis in Drosophila.

If Gyc76C functions as an ITP receptor in Drosophila, it should be expressed in cells and tissues that are innervated by ITPa/ITPL-expressing neurons, as well as in tissues, which mediate some of the known hormonal functions of ITP. To validate this prediction, we used a recently generated T2A-GAL4 knock-in line for Gyc76C (Figure 5A; Kondo et al., 2020) to comprehensively map its expression throughout larval Drosophila (Figure 5—figure supplement 1) and in adult males (Figure 5B–O) and females (Figure 5—figure supplement 2). In males, Gyc76C-T2A-GAL4 drives GFP expression throughout the adult intestinal tract, including the anterior midgut (Figure 5B), ureter (of renal tubules) (Figure 5C), and posterior midgut (Figure 5D). Importantly, in agreement with the role of Drosophila ITP in regulating osmotic (Gáliková et al., 2018) and metabolic homeostasis (Gáliková and Klepsatel, 2022), Gyc76C is highly expressed in the renal tubules (Figure 5E), rectum (Figure 5F), and adipocytes of the fat body (Figure 5G). Moreover, we see a convergence of ITPa-immunolabeled axon terminations and Gyc76C expression in the anterior midgut (Figure 5H) and the rectal papillae in the rectum (Figure 5I), the latter of which are important for water reabsorption as first proposed nearly a century ago (Wigglesworth, 1932). Gyc76C is also broadly expressed in neurons throughout the brain (Figure 5J) and VNC (Figure 5K). Consistent with the role of ITP in regulating circadian rhythms, Gyc76C is expressed in glia clock cells (Figure 5L), and subsets of dorsal clock neurons (labeled with antibody against the clock protein Period) that are near the axon terminations of the clock neurons LNd^ITP and 5^th-LN_v (Figure 5M). Gyc76C is not expressed in lateral clock neurons which are situated more closely to ITPa-expressing clock neurons (Figure 5L). Similar to males, Gyc76C-T2A-GAL4 also drives GFP expression in the female fat body, renal tubules, midgut, brain, VNC and subsets of dorsal clock neurons (Figure 5—figure supplement 2). Interestingly, Gyc76C is not expressed in male IPCs (labeled with antibody against DILP2) (Figure 5N) but is expressed in a subset of female IPCs (Figure 5—figure supplement 2). However, Gyc76C is not expressed in endocrine cells producing glucagon-like adipokinetic hormone (AKH) in either males (Figure 5O) or females (Figure 5—figure supplement 2). Interestingly, L-NSC^DH31, which innervate the corpora allata, might utilize both DH₃₁ and ITPa/ITPL1 to modulate juvenile hormone production since Gyc76C is expressed in the corpora allata (Figure 5O). Lastly, we also explored the distribution of Gyc76C in larval tissues, where expression was detected in the adipocytes of the fat body, all the regions of the gut and in renal tubules (Figure 5—figure supplement 1). Gyc76C is widely distributed in the larval nervous system, with high expression in the endocrine ring gland, where ITPa-immunoreactive axons terminate (Figure 5—figure supplement 1). Taken together, the cellular expression of Gyc76C in the nervous system and peripheral tissues of both larval and adult Drosophila further indicates that it could mediate the known effects of ITP.

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Gyc76C is necessary for ITPa-mediated inhibition of renal tubule secretion ex vivo

ITP has been shown to modulate osmotic homeostasis in Drosophila by suppressing excretion (Gáliková et al., 2018). While the precise mechanisms underlying the anti-diuretic effects of Drosophila ITP are not known, previous research in other systems provide important insights. For instance, in the locust Schistocerca gregaria, ITPa but not ITPL promotes ion and water reabsorption across the hindgut (Audsley et al., 1992a; Audsley et al., 1992b; King et al., 1999; Wang et al., 2000), thereby promoting anti-diuresis. Previous experiments have shown that the MTs are also targeted by anti-diuretic hormones: CAPA neuropeptides inhibit diuresis in some insects, including Drosophila via direct hormonal actions on the renal tubules (Paluzzi et al., 2008; MacMillan et al., 2018; Sajadi et al., 2020; Sajadi et al., 2023). Given the expression of Gyc76C, our candidate ITP receptor, in both the hindgut and renal tubules, ITP could modulate osmotic and/or ionic homeostasis by targeting these two excretory organs. Hence, we utilized the Ramsay assay (Figure 6A) to monitor ex vivo fluid secretion by MTs in response to application of recombinant ITPa. Interestingly, recombinant ITPa does not influence rates of secretion by unstimulated tubules (Figure 6B). Since the basal secretion rates are quite low, we tested if ITPa can inhibit secretion stimulated by LK, a diuretic hormone targeting stellate cells (O’Donnell et al., 1996), and a calcitonin-related peptide, DH₃₁, which acts on principal cells (Johnson et al., 2005). Recombinant ITPa inhibits LK-stimulated secretion by MTs from w¹¹¹⁸ flies (Figure 6C), indicating that stellate cell-driven diuresis is sensitive to this anti-diuretic hormone. Similarly, DH₃₁-stimulated secretion was also inhibited by recombinant ITPa (Figure 6D), demonstrating that principal cells are also modulated by ITPa. This result confirmed that the effect of ITPa on osmotic homeostasis are mediated, at least partially, via actions on renal tubules and that both major cell types are targeted.

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We next utilized the Ramsay assay to assess if Gyc76C is necessary for the inhibitory effects of ITPa on renal tubule secretion. Notably, knocking down expression of Gyc76C in stellate cells using c724-GAL4 abolished the anti-diuretic action of ITPa in LK-stimulated tubules (Figure 6E). Similarly, tubules in which Gyc76C was knocked down using the LK receptor GAL4 (Zandawala et al., 2018b) do not exhibit reduced secretion following ITPa application (Figure 6—figure supplement 1). Additionally, knocking down expression of Gyc76C in principal cells using uro-GAL4 abolished the anti-diuretic action of ITPa in DH₃₁-stimulated tubules (Figure 6F). Surprisingly, the application of ITPa alone promotes fluid secretion compared to unstimulated controls in tubules with Gyc76C knockdown (Figure 6E and F). This effect is more prominent in tubules with Gyc76C knockdown, specifically in the principal cells (Figure 6E and F). This suggests that ITPa could also interact with other yet unknown receptors in addition to Gyc76C. Nonetheless, these results indicate that ITPa exerts its anti-diuretic effects on MTs via Gyc76C, which acts as a functional ITPa receptor in both stellate and principal cells of the tubules.

ITPa activates Gyc76C in HEK293T cells

To further test whether ITPa activates Gyc76C, we leveraged a heterologous expression assay. HEK293T cells were transfected with Gyc76C-HA and/or the fluorescent protein-based cGMP indicator Green cGull (Matsuda et al., 2017) and subjected to live-cell imaging (Figure 7A). At 4 min after 50 nM, 250 nM, or 500 nM ITPa treatment, cells expressing HA-tagged Gyc76C and Green cGull exhibited 34%, 88.7%, and 111.4% increases in fluorescence intensity, respectively, indicating a dose-dependent increase in cGMP (Figure 7B-E, Figure 7—figure supplement 1). The same measurements in control cells transfected with only Green cGull failed to demonstrate a clear dose-dependent response with respective increases of 17.5%, 39.8%, and 28.2% (Figure 7, Figure 7—figure supplement 1). Area under the curve analysis confirmed significant differences between Gyc76C and control conditions (Figure 7G). Exogenous protein expression levels were confirmed with post-hoc staining of Gyc76C-HA and Green cGull (Figure 7—figure supplement 1). Several cells were found to express Green cGull independent of Gyc76C expression, explaining absence of strong response to ITPa in some cells. Finally, staining intensities were plotted against peak live-cell fluorescence increases, revealing weak negative correlations between exogenous protein expression and increases in cGMP signal (Figure 7—figure supplement 1). The observed correlations may reflect lower dynamic ranges due to higher baseline fluorescence in cells receiving more exogenous DNA. Taken together, these live-imaging results strongly suggest that ITPa can induce increased cGMP production through Gyc76C.

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ITPa-producing neurons are activated and release ITPa under desiccation

Having validated Gyc76C as a functional ITPa receptor, we next wanted to determine the context(s) during which ITP signaling is active in vivo. ITP expression has been shown to be upregulated during desiccation (Gáliková et al., 2018). However, whether this increased transcription is also coupled with increased ITP signaling is unknown. Consistent with its role as an anti-diuretic hormone, we hypothesized that ITP signaling is increased under desiccation. To test this hypothesis, we employed CaLexA (Masuyama et al., 2012), a transcriptional reporter of neuronal activity, to monitor the activity of L-NSC^ITP in flies exposed to different contexts that challenge their osmotic homeostasis. In agreement with our prediction, L-NSC^ITP are more active (indicated by increased GFP immunofluorescence) in both males (Figure 8A) and females (Figure 8B) that were desiccated compared to flies that were kept under normal conditions. Moreover, L-NSC^ITP activity returned to normal levels in rehydrated flies that were previously exposed to desiccation (Figure 8A and B). In order to confirm that increased neuronal activity translates into increased peptide release, we independently quantified ITPa immunofluorescence in different subsets of ITPa-producing brain neurons (Figure 8C and D). We observed reduced fluorescence in ITPa-expressing NSC as well as clock neurons in both males (Figure 8C) and females (Figure 8D) that had been exposed to desiccation stress. ITPa immunofluorescence returned to normal levels in desiccated flies that were then allowed to rehydrate. Since ITP mRNA is upregulated during desiccation, reduced immunofluorescence indicates increased release and not decreased peptide synthesis. Hence, not only do the L-NSC^ITP release ITPa into the circulation during desiccation, but the 5^th-LN_v and LN_d^ITP likely release ITPa within the brain to modulate other circuits during desiccation. Together, these results demonstrate that ITP neurons are activated and release ITPa during desiccation.

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ITP knockdown in ITP-RC-T2A-GAL4 cells impacts osmotic and metabolic homeostasis

Insect ITP is evolutionarily related to CHH, which, as its name indicates, regulates glucose homeostasis in crustaceans (Chen et al., 2020). A previous study employing ubiquitous ITP knockdown and overexpression suggests that Drosophila ITP also regulates feeding and metabolic homeostasis (Gáliková and Klepsatel, 2022) in addition to osmotic homeostasis (Gáliková et al., 2018). However, given the nature of the genetic manipulations (ectopic ITPa overexpression and knockdown of ITP in all tissues) utilized in those studies, it is difficult to parse the effects of ITP signaling from ITPa-producing neurons. To fill this gap and understand the role of ITP signaling in regulating osmotic and metabolic homeostasis in vivo, we specifically knocked down ITP using the ITP-RC-T2A-GAL4, which includes all the ITPa-expressing neurons. To avoid any developmental effects, we combined temperature-sensitive tubulin GAL80 with the GAL4 line (here referred to as ITP-RC-GAL4^TS) to restrict ITP knockdown specifically to the adult stage. For these experiments, both the ITP-RNAi and the control luciferase RNAi lines were first backcrossed for five generations into the wild-type background to minimize genetic background effects. We first successfully confirmed the effectiveness of ITP-RNAi by quantifying ITPa immunofluorescence in the brains of control and ITP knockdown flies (Figure 9A). In agreement with the anti-diuretic effects of ITPa ex vivo, ITP knockdown resulted in reduced desiccation tolerance (Figure 9B). This is likely a result of reduced water retention (Figure 9C) and increased defecation (Figure 9D). This reduced water content was also evident from the visibly shrunken abdomens of ITP knockdown females compared to controls (Figure 9E).

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Beyond its effects on osmotic balance, ITP knockdown in ITP-RC neurons also compromised metabolic homeostasis. Females with ITP knockdown exhibited reduced survival under starvation (Figure 9F), prompting us to assess their circulating and stored macronutrients. As expected based on lowered starvation tolerance, these flies displayed significantly lower circulating glucose (Figure 9G) and lipid stores in the fat body (Figure 9I and J). Glycogen levels, however, remained unaltered compared to controls (Figure 9H). As a consequence of these depleted energy reserves, the size of the ovaries was reduced in ITP knockdown females (Figure 9K). Surprisingly, the reduction in energy reserves was not attributable to decreased food intake. On the contrary, ITP knockdown flies consumed more food than controls (Figure 9L). Independently, we also assayed the food preference of flies when given a choice between a nutritive sugar and a sweeter non-nutritive sugar, since it can report deficits in mechanisms that monitor internal metabolic state. While fed flies of both the control and experimental genotypes showed no preference (Figure 9M), starved control flies exhibited a shift towards caloric nutritive sugars (Figure 9N), which reflects their drive to restore energy balance. In contrast, starved ITP knockdown flies did not display this shift in preference towards nutritive sugars (Figure 9N), indicating a disruption in the integration of internal metabolic cues with taste-driven food selection.

Collectively, these results demonstrate that disrupting ITP signaling from ITP-RC neurons leads to profound systemic effects on osmotic regulation, metabolic balance, and feeding behaviors, underscoring the pivotal role of ITP in coordinating diverse physiology and behaviors.

ITPa overexpression in ITP-RC-T2A-GAL4 cells modulates osmotic and metabolic homeostasis

The ITP-RNAi used here targets all ITP isoforms. Hence, the phenotypes observed following ITP knockdown cannot directly be attributed to ITPa. Therefore, we complemented these analyses by specifically overexpressing ITPa in adult females using ITP-RC-GAL4^TS (Figure 10). We first confirmed that driving UAS-ITPa with ITP-RC-GAL4^TS indeed results in increased ITPa peptide levels in L-NSC^ITP (Figure 10A). In agreement with our ex vivo secretion data, ITPa overexpression improves desiccation tolerance (Figure 10B), likely due to increased water retention, since flies overexpressing ITPa had higher body water content (Figure 10C) and bloated abdomens (Figure 10D). Independently, we also assessed recovery from chill-coma as an indirect measure of flies’ ionoregulatory capacity (MacMillan et al., 2012). ITPa overexpression had no impact on chill coma recovery and tolerance to salt stress (Figure 10—figure supplement 1). With regard to metabolic physiology, flies with ITPa overexpression survive longer under starvation (Figure 10E). These flies had reduced glucose levels (Figure 10F) but their glycogen levels were unaltered (Figure 10G). ITPa overexpression also led to increased lipid levels (Figure 10H). In addition, flies with ITPa overexpression showed defects in preference between nutritive versus non-nutritive sugar (Figure 10I and J) and had larger ovaries (Figure 10K). Thus, ITPa overexpression largely results in opposite phenotypes compared to those seen following ITP knockdown.

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Independently, since ITPa is released from both clock neurons and L-NSC^ITP during desiccation, we asked whether ITPa overexpression in both neuron types affected rhythmic locomotor activity under normal and desiccating conditions. We did not observe any drastic differences in locomotor activity of flies kept under normal conditions and subsequently transferred to empty vials (desiccation conditions) (Figure 10L). Therefore, the function of ITPa released from clock neurons during desiccation remains to be determined.

ITPa signals via Gyc76C in the renal tubules to modulate osmotic homeostasis

We next explored the role of ITP signaling via Gyc76C in maintaining osmotic homeostasis in vivo. In order to test if the effects on osmotic homeostasis were mediated via Gyc76C in the renal tubules, we monitored osmotic and ionic/salt stress tolerance of flies in which Gyc76C was specifically knocked down in MT principal or stellate cells using the uro-GAL4 and c724-GAL4, respectively (Figure 11). As expected, flies with Gyc76C knockdown in the MTs exhibit reduced tolerance to desiccation irrespective of the cell type, principal or stellate, being targeted (Figure 11A and D). Interestingly, salt stress impacted flies differently depending on the cell type in which Gyc76C was knocked down. Principal cell knockdown led to increased survival (Figure 11B), whereas stellate cell knockdown resulted in reduced survival (Figure 11E), which could reflect functional differences between the two cell types. Lastly, Gyc76C knockdown in either MT cell type increased the time taken to recover from chill-coma, highlighting deficits in the ability to maintain ionic homeostasis (Figure 11C and F). In summary, ITPa is released into the circulation during desiccation and modulates the MTs to promote tolerance to osmotic and ionic stresses. This evidence suggests that the hormonal effect of ITPa is likely mediated via Gyc76C expressed in the stellate and principal cells of the MTs.

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ITPa-Gyc76C signaling to the fat body influences metabolic physiology and associated behaviors

Having identified the inter-organ pathway via which ITPa modulates osmotic homeostasis, we next wanted to characterize the pathway regulating metabolic physiology. Since Gyc76C is expressed in the fat body and only the female IPCs, but not in AKH-producing cells, we hypothesized that ITP primarily regulates metabolic homeostasis via direct signaling to the fat body. To test this prediction, we specifically knocked down Gyc76C in the female fat body using yolk-GAL4. Flies with Gyc76C knockdown in the fat body exhibit a drastic reduction in starvation tolerance compared to control flies (Figure 12A). Remarkably, these flies start dying after only 4 hr of starvation, whereas control flies can normally tolerate at least 24 hr of starvation. Therefore, we investigated whether Gyc76C signaling to the fat body impacts energy stores. In agreement with reduced starvation survival, Gyc76C knockdown flies have lower hemolymph glucose (Figure 12B), unaltered glycogen levels (Figure 12C), and lower lipids in the fat body (Figure 12D and E) compared to controls. Hence, lower lipid levels, especially in the fat body, likely contribute to reduced starvation survival. Interestingly, the reduction in energy stores is not due to decreased food intake because Gyc76C knockdown flies fed more than controls (Figure 12F). In addition, these flies displayed altered food preferences. Specifically, they preferred yeast over sucrose (Figure 12G), possibly to mitigate protein or general caloric deficits since the protein in yeast yields greater caloric value than sugar. Independently, we also assayed the preference of flies for nutritive versus non-nutritive sugars. While there was no preference for nutritive or non-nutritive sugar in fed flies (Figure 12H), control flies showed increased preference for nutritive sugar during starvation (Figure 12I). Conversely, starved Gyc76C knockdown flies displayed a slight preference for non-nutritive sugar over nutritive sugar (Figure 12I), suggesting disrupted integration of taste signals with the internal metabolic state. In summary, these experiments indicate that Gyc76C signaling in the fat body is vital in regulating feeding, metabolic homeostasis, and consequently survival.

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Next, we examined if fat body-specific Gyc76C knockdown impacts other tissues and behaviors. We first observed that Gyc76C knockdown flies had drastically shrunken ovaries (Figure 12J). In addition, knockdown flies also defecated more than controls (Figure 12K) and relatedly had a lower water content (Figure 12L). These effects could either be caused by altered feeding and metabolism and/or via an indirect impact on insulin and ITP signaling amongst other pathways. Particularly, reduced insulin and ITP signaling could result in the observed reproductive and excretory phenotypes, respectively. Therefore, we quantified DILP2 and ITPa peptide levels in the brain NSC following knockdown of Gyc76C in the fat body. Indeed, knockdown flies have reduced DILP2 peptide levels (Figure 12M and O). However, we did not observe any differences in ITPa peptide levels in L-NSC^ITP (Figure 12N and O). These results suggest that the shrunken ovaries could be directly caused by reduced nutrient stores as well as via an indirect effect on insulin and possibly juvenile hormone signaling since L-NSC^DH31 innervate the corpora allata. The increased defecation, on the other hand, could likely be a consequence of increased feeding or due to an impact on another osmoregulatory pathway. To further substantiate the fat body-specific role of Gyc76C, we repeated several of the aforementioned analyses using an independent RNAi line (Gyc76C RNAi #2) and observed comparable phenotypes. Flies with Gyc76C knockdown in the fat body using Gyc76C RNAi #2 exhibited reduced tolerance to both desiccation and starvation stress (Figure 12—figure supplement 1). These flies also displayed reduced lipid stores and smaller ovaries (Figure 12—figure supplement 1). Our findings provide strong evidence that disruption of Gyc76C signaling in the fat body exerts profound systemic effects on multiple aspects of physiology and behavior.

Lastly, we monitored general locomotor activity and starvation-induced hyperactivity, the latter of which is largely governed by AKH, insulin and octopamine signaling (Lee and Park, 2004; Yu et al., 2016a; Pauls et al., 2021). Flies with Gyc76C knockdown in the fat body displayed reduced daytime activity when kept under either fed or starved conditions for one day (Figure 12P–R). Hence, Gyc76C signaling in the fat body does not appear to impact starvation-induced hyperactivity. However, the effect on general locomotor activity led us to examine the activity of fed flies in more detail over a longer time course. For this, we monitored the activity of flies for 10 days under 12:12 hr light/dark cycles and a subsequent 10 days under constant darkness (Figure 12—figure supplement 2). While Gyc76C knockdown flies displayed reduced activity on day 1, the average activity of these flies over days 2–6 was not significantly different from the controls (Figure 12—figure supplement 2). Interestingly, flies with Gyc76C knockdown in the fat body appeared to be more sensitive to differences in light cues, showing a strong reduction in locomotor activity only when switched to constant darkness from 12:12 hr light-dark cycles (Figure 12—figure supplement 2).

In conclusion, the phenotypes seen following Gyc76C knockdown in the fat body largely mirror those seen following ITP knockdown in ITP-RC neurons, providing further support that ITPa mediates its effects via Gyc76C.

Synaptic and peptidergic connectivity of ITP neurons

After characterizing the functions of ITP signaling to the renal tubules and the fat body, we wanted to identify pathways regulating ITP signaling and its downstream neuronal targets. To address this, we took advantage of the recently completed FlyWire adult brain connectome (Dorkenwald et al., 2024; Schlegel et al., 2024) to identify pre- and post-synaptic partners of ITP neurons. ITP neurons have a characteristic morphology which was used to identify them in the connectome (Figure 13A; McKim et al., 2024; Reinhard et al., 2024). LN_d^ITP and 5^th-LN_v displayed numerous input and output synapses in the brain (Figure 13B, Figure 13—figure supplement 1). In particular, these neurons have extensive synaptic output in the superior lateral protocerebrum, where Gyc76C-expressing dorsal clock neurons reside (Figure 5M, Figure 5—figure supplement 2). Consistent with the high number of synapses, both LN_d^ITP and 5^th-LN_v receive inputs and provide outputs to a broad range of neurons (Figure 13C and D). In addition, both cell types are upstream of at least one pair of DH₃₁-expressing NSC (L-NSC^DH31) (Figure 13D). However, it is not yet clear whether these NSCs are the same ones as the L-NSCs^DH31 that co-express ITPa (Figure 1B and E), since there are three pairs of L-NSC^DH31 in the adult brain (Reinhard et al., 2024). Therefore, we did not examine the synaptic connectivity of L-NSC^DH31 here. Interestingly, LN_d^ITP receive indirect inputs from VP1l thermo/hygrosensory neurons (Figure 13E; Choi et al., 2022). LN_d^ITP co-express neuropeptide F (NPF) and could be the same NPF-expressing neurons which have recently been implicated in water seeking during thirst (Ramirez et al., 2025). This connectivity could also explain why LN_d^ITP show reduced ITPa immunolabeling (indicating ITPa release) following desiccation (Figure 8C and D).

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In contrast to LN_d^ITP and 5^th-LN_v clock neurons, L-NSC^ITP form few significant synaptic connections within this brain volume (Figure 13B, D, Figure 13—figure supplement 1). Since these neurons are neurosecretory in nature, their peptides are released from axon terminations in neurohemal areas outside the brain, where regulatory inputs could be located (Dircksen et al., 2008; Kahsai et al., 2010; McKim et al., 2024). It is worth noting that L-NSC^ITP output onto other NSC subtypes which secrete osmoregulatory hormones like DH₃₁ and corazonin (CRZ) (Figure 13F). DH₃₁ is a diuretic hormone, whereas CRZ can inhibit CAPA, another diuretic hormone (Zandawala et al., 2021).

Since L-NSC^ITP receive few synaptic inputs, we hypothesized that their activity, especially during desiccation, is regulated either by cell-autonomous osmosensing or by paracrine and hormonal modulators which transmit the signal from other central or peripheral osmosensors. To address this, we mined single-cell transcriptomes of different subsets of ITP-expressing neurons (Figure 13G, Figure 13—figure supplement 2) from whole brain and VNC datasets (Davie et al., 2018; Allen et al., 2020) based on markers identified here and previously (Kahsai et al., 2010; Reinhard et al., 2024). To assess if ITP-expressing neurons are cell autonomously osmosensitive, we first examined the expression of transient receptor potential (TRP) and pickpocket (ppk) channels which have been shown to confer osmosensitivity to cells (Sharif-Naeini et al., 2008; Cameron et al., 2010). Although Trpm, a TRP channel, was expressed in LN^ITP (Figure 13G), we did not detect expression of any TRP or ppk channels in L-NSC^ITP and L-NSC^DH31 (not shown). Thus, unless other (non-characterized) osmosensors are expressed in L-NSC^ITP, the internal state of thirst/desiccation is likely conveyed to L-NSC^ITP via neuromodulators. Intriguingly, the dopamine receptor, Dop2R, is highly expressed in all ITP neuron subtypes (Figure 13H, Figure 13—figure supplement 2). Compared to the ITP-expressing neurons in the VNC, the brain neurons seem to be extensively modulated by different neuropeptides (Figure 13I, Figure 13—figure supplement 2), including those that regulate osmotic homeostasis (diuretic hormone 44, LK, and DH₃₁), and feeding and metabolic homeostasis (insulin, Ast-A, CCAP, drosulfakinin, and sNPF). Lastly, with the exception of L-NSC^ITP, all ITP neurons express high levels of neurotransmitter receptors (Figure 13J, Figure 13—figure supplement 2). This is consistent with fewer synaptic inputs to L-NSC^ITP. In conclusion, the ITP neuron connectomes and transcriptomes provide the basis to functionally characterize signaling pathways regulating ITP signaling in Drosophila.

Insect ITPs are members of the multifunctional family of CHH/MIH neuropeptides that have been intensely investigated in crustaceans for their role in development, reproduction, and metabolism (Webster et al., 2012). In Drosophila, the three ITP isoforms (ITPa, ITPL1, and ITPL2) were until very recently among the very few neuropeptides whose receptors had not been identified. However, recently an ITPL2-activated GPCR, TkR99D, was identified (Xu et al., 2023) similar to the ITPL-activated BNGR-A24 in the moth Bombyx (Nagai et al., 2014). Thus, receptors for Drosophila ITPa and ITPL1 remained to be identified. Furthermore, the neuronal pathways and functional roles of the three ITP isoforms have remained relatively uncharted. Here, using a multipronged approach consisting of anatomical mapping, single-cell transcriptomics, in vitro tests of recombinant ITPa and genetic experiments in vivo, we comprehensively mapped the tissue expression of all three ITP isoforms and revealed roles of ITP signaling in regulation of osmotic and metabolic homeostasis via action on MTs and fat body, respectively. We furthermore identified and functionally characterized a receptor for the amidated isoform ITPa, namely the mGC Gyc76C and analyzed its tissue distribution and role in systemic homeostasis. Lastly, we performed connectomics and single-cell transcriptomic analyses to identify synaptic and paracrine pathways upstream and downstream of ITP-expressing neurons. Together, our systematic characterization of ITP signaling establishes a tractable system to decipher how a small set of neurons integrates diverse inputs and orchestrates systemic homeostasis in Drosophila (Figure 14).

Figure 14

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Custom code used for connectome and single-cell transcriptome analyses is available at: https://github.com/Zandawala-lab/Gera-et-al-2025-Drosophila-ITPa-Gyc76C, copy archived at Zandawala and McKim, 2025. Custom code and output files are available at: https://doi.org/10.5281/zenodo.16747141.

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Author details

1. Jayati Gera

   Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, Julius-Maximilians-University of Würzburg, Am Hubland, Würzburg, Germany

   Contribution

   Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4744-1546

2. Marishia Agard

   Department of Biology, York University, Toronto, Canada

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Hannah Nave and Austin B Baldridge

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0009-1463-6409

3. Hannah Nave

   Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, Julius-Maximilians-University of Würzburg, Am Hubland, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing, Supervision

   Contributed equally with

   Marishia Agard and Austin B Baldridge

   Competing interests

   No competing interests declared

4. Austin B Baldridge

   Department of Biochemistry and Molecular Biology, University of Nevada, Reno, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Marishia Agard and Hannah Nave

   Competing interests

   No competing interests declared

5. Farwa Sajadi

   Department of Biology, York University, Toronto, Canada

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Leena Thorat

   Department of Biology, York University, Toronto, Canada

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

7. Theresa H McKim

   1. Department of Biology, University of Nevada, Reno, United States
   2. Integrative Neuroscience Program, University of Nevada, Reno, United States

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing – review and editing, Computational analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8501-6487

8. Shu Kondo

   Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan

   Contribution

   Resources, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4625-8379

9. Dick R Nässel

   Department of Zoology, Stockholm University, Stockholm, Sweden

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1147-7766

10. Mitchell H Omar

    1. Department of Biochemistry and Molecular Biology, University of Nevada, Reno, United States
    2. Integrative Neuroscience Program, University of Nevada, Reno, United States

    Contribution

    Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1713-4473

11. Jean-Paul Paluzzi

    Department of Biology, York University, Toronto, Canada

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7761-0590

12. Meet Zandawala

    1. Neurobiology and Genetics, Theodor-Boveri-Institute, Biocenter, Julius-Maximilians-University of Würzburg, Am Hubland, Würzburg, Germany
    2. Department of Biochemistry and Molecular Biology, University of Nevada, Reno, United States
    3. Integrative Neuroscience Program, University of Nevada, Reno, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    mzandawala@unr.edu

    Competing interests

    Reviewing editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0001-6498-2208

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Citations by DOI

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  citations for umbrella DOI https://doi.org/10.7554/eLife.97043

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  citations for Reviewed Preprint v1 https://doi.org/10.7554/eLife.97043.1