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

Abstract

Insects have evolved a variety of neurohormones that enable them to maintain nutrient and osmotic homeostasis. Here, we characterized the ion transport peptide (ITP) signaling system in Drosophila. The Drosophila ITP gene can generate three different peptide isoforms: ITP amidated (ITPa) and two ITP-like (ITPL1 and ITPL2) isoforms. We comprehensively characterized the expression of all three ITP isoforms in the nervous system and peripheral tissues. Our analyses reveal widespread expression of ITP isoforms. Moreover, we show that ITPa-producing neurons are activated and release ITPa during dehydration. Furthermore, recombinant Drosophila ITPa inhibits diuretic peptide-induced renal tubule secretion ex vivo, thus confirming its role as an anti-diuretic hormone. Using a phylogenetic-driven approach, an ex vivo secretion assay and a heterologous mammalian cell-based assay, we identified and functionally characterized Gyc76C, a membrane guanylate cyclase, as a bona fide Drosophila ITPa receptor. Extensive anatomical mapping of Gyc76C reveals that it is highly expressed in larval and adult tissues associated with osmoregulation (renal tubules and rectum) and metabolic homeostasis (fat body). Consistent with this expression, knockdown of Gyc76C in renal tubules impacts tolerance to osmotic and ionic stresses, whereas knockdown specifically in the fat body impacts feeding, nutrient homeostasis, and associated behaviors. We also complement receptor knockdown experiments with ITP knockdown and ITPa overexpression in ITPa-producing neurons. Lastly, we utilized connectomics and single-cell transcriptomics to identify pathways via which ITP neurons integrate hygrosensory inputs and interact with other homeostatic hormonal pathways. Taken together, our systematic characterization of ITP signaling establishes a tractable system to decipher how a small set of neurons integrates diverse inputs to orchestrate systemic homeostasis in Drosophila.

Keywords: D. melanogaster; atrial natriuretic peptide; computational biology; connectomics; endocrine signaling; metabolism; neuropeptide; neuroscience; osmoregulation; systems biology.

Figure 1.. Ion transport peptide (ITP) splicing pattern and expression of ITP transcript variants in the nervous system of adult male Drosophila.

(A) Drosophila ITP gene can generate 5 transcript variants (ITP-RC, RD, RE, RF, and RG). ITP-RC encodes ITPL1 precursor, ITP-RD, RF, and RG all encode ITPL2 precursor, and ITP-RE encodes a precursor that produces the amidated ITP (ITPa) peptide. Gray boxes represent exons and lines represent introns (drawn to scale). The regions encoding the open reading frame are colored (pink, green or blue). ITP is located on the second chromosome and numbers on the top indicate the genomic location. ITP-RC-T2A-GAL4 drives GFP (UAS-JFRC81GFP) expression in the (B) brain and (C and D) ventral nerve cord (VNC). (B’’) shows another brain preparation (same as in Figure 2) where axons of ITP-RC neurons are clearly visible. All images are from male flies. Within the brain, ITP-RC is co-expressed with ITPa in four pairs of lateral neurosecretory cells (L-NSC^ITP), one pair of diuretic hormone 31 (DH₃₁)-expressing lateral neurosecretory cells (L-NSC^DH31), one pair of fifth ventrolateral neurons (5^th-LN_v) and one pair of dorsolateral neurons (LN_d^ITP). L-NSC^ITP and L-NSC^DH31 are a subset of lateral neuroendocrine cells and the single pairs of 5^th-LN_v and LN_d^ITP belong to the circadian clock network. Within the VNC, ITP-RC is co-expressed with ITPa in abdominal ganglion neurons (iag), which innervate the rectal pad. In addition, ITP-RC is expressed in a pair of Tv* neurons near the midline in each thoracic neuromere. These neurons are located next to the FMRFamide-expressing Tv neurons (see Figure 2). ITP-RD-T2A-GAL4 also drives GFP expression in the (E and F) brain and (G and H) VNC. ITP-RD is expressed in L-NSC^ITP, 5^th-LN_v, and LN_d^ITP neurons, as well as glia. Within the VNC, ITP-RD is expressed in neurons which are not iag or Tv* neurons. (I) Summary of ITP isoform expression within the nervous system. Gray box indicates presence and white box indicates absence.

Figure 2.. Ion transport peptide (ITP) is co-expressed with other neuropeptides in the nervous system of adult male Drosophila.

(A) A single pair of ITP-RC>GFP-positive lateral neurosecretory cells in the dorsal brain (marked by an arrowhead) co-express diuretic hormone 31 (DH₃₁). (B) ITP-RC drives GFP expression in a pair of Tv* neurons near the midline in each thoracic neuromere. These neurons are located next to the FMRFamide-expressing Tv neurons. (C and D) ITP-RC and ITPa-expressing peripheral neurons (marked by asterisk) and abdominal ganglion neurons (marked by arrowheads) co-express allatostatin-A (Ast-A) neuropeptide. (E) CCAP>GFP-positive neurons co-express ITPa (and Ast-A by extension) in the abdominal ganglion neurons (marked by arrowheads). (F) ITP-RC and ITPa-expressing neurons are distinct from Ast-A-expressing neurons in the brain. (G) Schematic of the nervous system showing neuropeptides (transcripts or mature peptides) expressed in ITP neurons. Peripheral neurons on one side are marked by arrowheads. Based on previous reports (Kahsai et al., 2010; Hermann-Luibl et al., 2014; Zandawala et al., 2018a) and the present study.

Figure 3.. Ion transport peptide (ITP) expression in peripheral tissues of adult male Drosophila.

(A) Schematic showing the location of tissues where ITP is expressed. Created with BioRender.com. (B) t-SNE visualization of single-cell transcriptomes showing ITP expression in different tissues of adult Drosophila. ITP is broadly expressed in peripheral tissues, including (C) trachea, Malpighian tubules (tubule), body, (D) heart, fat body, and gut. ITP-RC-T2A-GAL4 drives GFP (UAS-JFRC81GFP) expression in (E) peripheral neurons with axons innervating the heart and (F) abdominal ganglion neurons which innervate the rectum. ITP-RC is not expressed in (G) the fat body, (H) midgut, or (I) Malpighian tubules. ITPa immunolabeling is present in (J) a pair of peripheral neurons (cell bodies marked by arrowheads) innervating the heart and (K) in abdominal ganglion neurons which innervate the rectum, but (L) absent in the midgut. ITP-RD-T2A-GAL4 drives GFP expression in (M) the heart and nephrocytes (marked by asterisk), (N) middle midgut, (O) posterior midgut, (P) ureter, and (Q) trachea. (R) ITP-RD is not expressed in the fat body.

Figure 4.. Ion transport peptide (ITP) signaling components are found in protostomes.

(A) Multiple sequence alignment of ITP precursor sequences. ITP is homologous to crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH). Note the conservation of six cysteine residues (highlighted in red) across all the species. C-terminal glycine, which is predicted to undergo amidation is colored in green. Species abbreviations: Drome, Drosophila melanogaster; Locmi, Locusta migratoria; Dapma, Daphnia magna; Carma, Carcinus maenas; Ixosc, Ixodes scapularis; Caeel, Caenorhabditis elegans; Hypdu, Hypsibius dujardini; Prica, Priapulus caudatus; Chala, Charonia lampas. (B) Maximum-likelihood phylogeny of membrane guanylate cyclase receptors identifies two clades that are restricted to protostome phyla which also have ITP. The clade containing D. melanogaster Gyc76C receptor are the putative ITPa receptors. Bootstrap values higher than 200 (based on 500 replicates) are indicated adjacent to the nodes. Drosophila guanylate cyclase alpha and beta subunits were used as outgroups.

Figure 4—figure supplement 1.. Drosophila orthologs of Bombyx mori ion transport peptide (ITP) and ITPL receptors.

(A) Drosophila CG8784 (PK2-R1), CG30340 and CG7887 (TkR99D) are orthologs of Bombyx mori BNGR-A2, BNGR-A34 and BNGR-A24, respectively. Ligands of these receptors based on previous studies are indicated (Nagai et al., 2014; Nagai-Okatani et al., 2016). (B) PK2-R1 is not expressed in the rectal papillae but is expressed in the neurons innervating the rectum. (C) CG30340 is not expressed in the rectum. (D) Tkr99D is also only expressed in axons innervating the rectum. (E) PK2-R1, (F) CG30340 and (G) TkR99D are all expressed in the midgut or the neurons innervating it. (H) PK2-R1, CG30340, and TkR99D are not expressed in the fat body but receptors for insulin (InR) and adipokinetic hormone (AkhR) are. (I) PK2-R1 and CG30340 are not expressed in Malpighian tubules. TkR99D and leucokinin receptor (Lkr) are expressed in stellate cells and diuretic hormone 31 receptor (Dh31-R) is expressed in principal cells. (J) Drosophila tachykinin 1 (DTK-1) but not ITPa activates TkR99D expressed in CHO-K1 cells stably expressing aequorin (CHOK1-aeq), a calcium-activated bioluminescent protein. (K) Pyrokinin 2 (PK2) but not ITPa activates PK2-R1 expressed in CHOK1-aeq cells. For (J and K), bars labeled with different letters are significantly different from each other (p<0.05, as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test).

Figure 4—figure supplement 2.. Expression of candidate ITP amidated (ITPa)/ITPL receptors in adult male Drosophila tissues.

(A) Gyc76C expression in Malpighian tubules (blue outline) is higher compared to Gyc32E. Gyc32E-GAL4 does not drive GFP expression in the (B) Malpighian tubules and (C) rectal pad but is expressed in the hindgut (marked by an asterisk) and (D) fat body of males. (E) Gyc32E-GAL4 drives nuclear mCherry expression in a subset of insulin-producing cells (labeled by DILP2 antibody).

Figure 5.. Gyc76C expression in adult male Drosophila.

(A) Schematic showing the generation of Gyc76C-T2A-GAL4 knock-in line. Gyc76C-T2A-GAL4 drives GFP (UAS-JFRC81GFP) expression in the (B) anterior midgut, (C) ureter, (D) posterior midgut, (E) Malpighian tubules, (F) ileum, rectum, (G) and adipocytes in the fat body. Gyc76C is expressed in the regions of (H) the anterior midgut and (I) rectal papillae in the rectum that are innervated by ITP amidated (ITPa)-expressing neurons. Gyc76C is also broadly expressed in the (J) brain and (K) ventral nerve cord. (L) Gyc76C is expressed in glial clock cells and (M) subsets of dorsal clock neurons (both labeled by Period antibody and marked by arrowheads). Gyc76C is not expressed in (N) insulin-producing cells (labeled by DILP2 antibody) and (O) adipokinetic hormone (AKH) producing endocrine cells but is expressed in the corpora allata (CA) (marked in white).

Figure 5—figure supplement 1.. Gyc76C expression in larval Drosophila.

Gyc76C-T2A-GAL4 drives GFP (UAS-JFRC81GFP) expression in the (A) fat body, (B) proventriculus, (C) anterior midgut, (D) middle midgut, (E) posterior midgut, (F) ureter, (G) Malpighian tubules, and (H) hindgut. Lipid droplets (magenta in panel A) were stained using Nile red. (I) Gyc76C is broadly expressed in the larval nervous system, including the ring gland, which is innervated by ITP amidated (ITPa)-expressing neurons (magenta).

Figure 5—figure supplement 2.. Gyc76c expression in adult female Drosophila.

Gyc76C-T2A-GAL4 drives GFP (UAS-JFRC81GFP) expression in the (A) fat body, (B) Malpighian tubules, and (C) the midgut. Gyc76C-T2A-GAL4 drives nuclear mCherry expression in the brain (D) and ventral nerve cord (E). (F and G) Gyc76C is expressed in subsets of clock neurons (labeled by Period antibody and marked by arrowheads). Gyc76C is expressed in (H) a subset of insulin-producing cells (labeled by DILP2 antibody) but not (I) in the adipokinetic hormone (AKH) producing endocrine cells.

Figure 6.. Recombinant Drosophila ITP amidated (ITPa) inhibits Malpighian tubule secretion via Gyc76C.

(A) Schematic of Ramsay assay used to monitor ex vivo secretion by tubules. (B) Application of Drosophila 500 nM ITPa does not affect basal secretion rates by unstimulated tubules. 500 nM ITPa inhibits both (C) 10 nM leucokinin (LK)-stimulated and (D) 1 μM diuretic hormone 31 (DH₃₁)-stimulated secretion rates. Importantly, while 500 nM ITPa inhibits (E) 10 nM LK-stimulated secretion and (F) 1 μM DH31-stimulated by renal tubules from control flies, this inhibitory effect is abolished in tubules where Gyc76C has been knocked down with UAS-Gyc76C RNAi (#106525) in stellate cells using the c724-GAL4 and in principal cells using uro-GAL4. Male Malpighian tubules were used for all experiments. For (B-D) p<0.05 and ****p<0.0001 as assessed by unpaired t-test. For (E and F), within each genotype, different letters denote secretion rates that are significantly different from one another (p<0.05) as assessed by two-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 6—figure supplement 1.. Recombinant Drosophila ITP amidated (ITPa) inhibits Malpighian tubule secretion via Gyc76C.

Recombinant ITPa expressed in AtT-20 cells inhibits leucokinin (LK)-stimulated secretion by renal tubules. This inhibitory effect is abolished in tubules where Gyc76C has been knocked down with UAS-Gyc76C RNAi (#106525) using the LK receptor GAL4 (Lkr-GAL4). Differences between treatments within and across genotypes are denoted by different letters as determined by two-way ANOVA followed by Tukey’s multiple comparisons test (p<0.05).

Figure 6—figure supplement 2.. Western blot analysis of recombinant ITP amidated (ITPa) produced in AtT-20 cells.

A single band corresponding to the molecular weight of ITPa (~9 kDa) is observed in lysates from AtT-20 cells expressing ITPa but not mCherry.

Figure 7.. ITP amidated (ITPa) activates Gyc76C heterologously expressed in HEK293T cells.

(A) Schematic of the heterologous assay used to functionally characterize Gyc76C. Created with BioRender.com. Application of (B) 50 mM, (C) 250 mM, or (D) 500 nM Drosophila ITPa to HEK293T cells transiently expressing Green cGull (cGMP sensor) and Gyc76C results in a dose-dependent increase in fluorescence compared to control cells which do not express Gyc76C. Graphs represent the mean fluorescence of 17–18 cells. Representative images showing fluorescence in (E) HEK293T cells expressing Gyc76C and (F) those without Gyc76C before and 4 min after the addition of 500 nm ITPa. (G) Area under the curve analysis demonstrates significant differences in Green cGull fluorescence increases between experimental and control conditions; *p<0.05 and ****p<0.0001 as assessed by nonparametric one-way ANOVA followed by Dunn’s test for multiple comparisons.

Figure 7—figure supplement 1.. ITP amidated (ITPa) activates Gyc76C in HEK293T cells.

Application of (A) 50 nm, (B) 250 nm, and (C) 500 nM Drosophila ITPa to HEK293T cells transiently expressing Green cGull (cGMP sensor) and Gyc76C (green) results in an increase in fluorescence compared to control cells (gray) which do not express Gyc76C. The graph represents the fluorescent intensities of individual cells over time. (D) Post-hoc immunohistochemistry of tested HEK293T cells (500 nM ITPa) reveals that not all cGull-expressing cells express Gyc76C-HA, explaining the lack of response to ITPa in some cells. (E and F) Graphs depicting stained Gyc76C-HA (E) and Green cGull (F) fluorescence intensities plotted against peak live Green cGull fluorescence increases (500 nM condition) indicate weak negative correlations between exogenous protein expression level and change in biosensor fluorescence.

Figure 8.. ITP amidated (ITPa) neurons are active and release ITPa during desiccation.

GFP immunofluorescence, indicative of calcium levels and measured using the CaLexA reporter, is increased in L-NSC^ITP of (A) male and (B) female flies exposed to desiccation. The GFP intensity returns to control levels in flies that were rehydrated following desiccation. ITPa immunofluorescence, indicative of peptide levels, is lowered in 5^th-LN_v, LN_d^ITP and L-NSC^ITP of (C) male and (D) female flies exposed to desiccation. ITPa peptide levels recover to control levels in flies that were rehydrated following desiccation. Lower peptide levels during desiccation indicate increased release. For all panels, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 9.. Knockdown of ion transport peptide (ITP) in adult female Drosophila impacts metabolic homeostasis, feeding, and associated behaviors.

(A) ITPa immunofluorescence is reduced in the L-NSC^ITP neurons of flies in which ITP was knocked down using ITP-RC-GAL4^TS (ITP-RC-T2A-GAL4 combined with temperature-sensitive tubulin-GAL80). Flies with ITP knockdown are (B) less resistant to desiccation tolerance, (C) have reduced water content, (D) increased defecation, and (E) shrunken abdomen. ITP knockdown flies (F) survive less under starvation, (G) have lower levels of circulating glucose, and (H) unaffected glycogen levels. However, reduced ITP signaling results in (I and J) less lipid levels (TAG = triacylglyceride), and (K) smaller ovaries. Moreover, ITP knockdown flies exhibit (L) increased feeding (over 24 hr) and (M and N) defects in preference for nutritive sugars when starved for 18 hr prior to testing. Abbreviations: Luc Ri, luciferase RNAi; ITP Ri, ITP RNAi. For (B and F), **p<0.01, ***p<0.001, as assessed by Log-rank (Mantel-Cox) test. For all others, *p<0.05, **p<0.01, and ****p<0.0001 as assessed by unpaired t-test.

Figure 10.. ITP amidated (ITPa) overexpression in adult female Drosophila impacts osmotic and metabolic homeostasis, feeding, and related behaviors.

(A) Overexpression of ITPa using ITP-RC-GAL4^TS results in increased ITPa immunofluorescence in L-NSC^ITP. ITPa overexpression results in (B) increased desiccation tolerance, (C) increased water content, and (D) a slightly bloated abdomen (marked by an asterisk). ITPa overexpression causes (E) increased starvation tolerance, (F) reduced circulating glucose levels but has no effect on (G) glycogen levels. (H) The size of neutral lipid droplets (stained with Nile red) is increased in flies with ITPa overexpression. (I and J) These flies also exhibit defects in preference for nutritive sugars when starved for 16 hr prior to testing. (K) ITPa overexpression flies have enlarged ovaries. (L) ITPa overexpression has no effect on locomotor activity under fed or desiccating conditions. Black bars indicate night-time and yellow bars indicate daytime. All experiments were performed at 29 °C. For (B and E), ****p<0.0001, as assessed by Log-rank (Mantel-Cox) test. For (A), *p<0.05 as assessed by unpaired t test. For all other experiments, *p<0.05, ***p<0.001, ****p<0.0001 as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. For clarity, significant pairwise differences compared to only the experimental treatment are indicated.

Figure 10—figure supplement 1.. ITP amidated (ITPa) overexpression in adult female Drosophila has no effect on cold and ionic stress.

Overexpression of ITPa using ITP-RC-GAL4^TS has no impact on (A) recovery from chill coma and (B) tolerance to salt stress, as assessed by Log-rank (Mantel-Cox) test.

Figure 11.. Female Malpighian tubule-specific knockdown of Gyc76C impacts osmotic and ionic homeostasis.

Knockdown of Gyc76C in both the (A) principal cells of renal tubules using uro-GAL4 and (D) stellate cells using c724-GAL4 reduces desiccation tolerance. Gyc76C knockdown in (B) principal cells increases survival under salt stress, whereas knockdown in (E) stellate cells lowers survival. (C and F) Gyc76C knockdown in principal or stellate cells increases the time taken for recovery from chill-coma. Abbreviation: Gyc76C Ri, Gyc76C RNAi. For all panels, **p<0.01, ****p<0.0001, as assessed by Log-rank (Mantel-Cox) test.

Figure 12.. Gyc76C knockdown in the female fat body using yolk-GAL4 impacts metabolic homeostasis, feeding, and associated behaviors.

Flies with fat body-specific Gyc76C knockdown with UAS-Gyc76C RNAi (#106525) are (A) extremely susceptible to starvation and (B) have reduced glucose levels. (C) Glycogen levels are unaltered in flies with fat body-specific Gyc76C knockdown. (D and E) However, lipid levels (TAG = triacylglyceride) are drastically reduced. Gyc76C knockdown flies exhibit (F) increased feeding (over 24 hr), (G) a preference for yeast over sucrose, and (H and I) defects in preference for nutritive sugars when starved for 4 hr prior to testing. Flies with Gyc76C knockdown in the fat body have (J) smaller ovaries, they (K) defecate more and have (L) reduced water content than the controls. For K, the number of excreta counted over 2 hr. Gyc76C knockdown also impacts (M) DILP2 peptide levels (N) but not ITP amidated (ITPa) levels in the neurosecretory cells. CTCF=Corrected Total Cell Fluorescence. (O) Representative confocal stacks showing DILP2 and ITPa immunostaining. Gyc76C knockdown flies also display reduced daytime locomotor activity under (P) fed and (Q) and starved conditions compared to controls. Black bars indicate nighttime and yellow bars indicate daytime. (R) Average night and daytime activity over one day under fed and starved conditions. For (A), ****p<0.0001, as assessed by Log-rank (Mantel-Cox) test. For (M and N), *p<0.05 as assessed by unpaired t test. For all others, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. For clarity, significant pairwise differences compared to only the experimental treatment are indicated.

Figure 12—figure supplement 1.. Gyc76C knockdown with an independent RNAi in females using yolk-GAL4 impacts stress tolerance, energy stores, and reproductive physiology.

Flies with fat body-specific Gyc76C knockdown using UAS-Gyc76C RNAi #2 (#57315) are (A) extremely susceptible to desiccation as well as (B) starvation. Furthermore, flies with Gyc76C knockdown in the fat body have (C) reduced lipid levels (Nile Red), and (D) smaller ovaries. Abbreviation: Gyc76C Ri #2, Gyc76C RNAi #2. For (A and B), ****p<0.0001, as assessed by Log-rank (Mantel-Cox) test.

Figure 12—figure supplement 2.. Gyc76C knockdown with UAS-Gyc76C Ri in the female fat body impacts general locomotor activity.

(A) Representative actograms (double-plotted) showing locomotor activity over 20 days. Yellow shading indicates light and gray shading indicates dark conditions. (B) Daily total activity of flies over 20 days. Gyc76C knockdown flies have reduced locomotor activity on the first day as well as under constant darkness. **p<0.01 and ****p<0.0001 as assessed by repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Average activity profiles over days 2–6. (D) Average night- and day-time activities between days 2–6 were not significantly different. Abbreviation: Gyc76C Ri, Gyc76C RNAi.

Figure 13.. Inputs and outputs of ion transport peptide (ITP) neurons based on connectomics and single-cell transcriptomics.

(A) Reconstruction of ITP amidated (ITPa)-expressing neurons using the complete electron microscopy volume of the adult female brain (data retrieved from the FlyWire platform). Four pairs of lateral neurosecretory cells (L-NSC^ITP) are gray, fifth ventrolateral neurons (5^th-LN_v) are cyan, and dorsolateral neurons (LN_d^ITP) are yellow. Diuretic hormone 31 (DH₃₁)-expressing lateral neurosecretory cells (L-NSC^DH31) are not shown since it is unclear which of the three pairs of L-NSC^DH31 co-expresses ITPa. (B) Location of input and output synapses are colored according to the ITP neuron type. (C) Proportion of input synapses (grouped by super class annotations for the FlyWire connectome Schlegel et al., 2024) to each ITP neuron type. (D) Reconstructions of neurons from different super classes providing inputs to (left) and receiving outputs from (right) 5^th-LN_v, LN_d^ITP, and L-NSC^ITP. Only the top 10 cell types are shown here. (Middle) Number of neurons, categorized by super class, providing inputs to and receiving outputs from 5^th-LN_v, LN_d^ITP, and L-NSC^ITP. (E) Thermo/hygrosensory input pathway to LN_d^ITP. (F) Output from L-NSC^ITP to other osmoregulatory hormone-producing cells. (G) Identification of single-cell transcriptomes representing different subsets of ITPa-expressing neurons in the adult brain dataset (Davie et al., 2018). Since both the 5^th-LN_v and LN_d^ITP co-express ITP, cryptochrome (cry), and neuropeptide F (NPF), these cells are grouped as LN^ITP. All three sets of neurons express genes required for neuropeptide processing and release (amon, svr, Pal2, Phm, and Cadps) and were identified based on the neuropeptides (ITP, NPF, Dh31, sNPF, and Tk) they express. Dot plots showing expression of (H) monoamine, (I) neuropeptide, and (J) neurotransmitter receptors in different sets of ITPa neurons.

Figure 13—figure supplement 1.. Input and output synapses of ion transport peptide (ITP) neurons.

(A) Input and (B) output synapses of L-NSC^ITP (gray), LN_d^ITP (yellow) and 5^th-LN_v (cyan). Higher magnification of images shown in Figure 13B. All synapses, including those not contributing towards significant connections (less than five synapses per connection), are shown here.

Figure 13—figure supplement 2.. Single-cell transcriptomes of ion transport peptide (ITP) neurons in the ventral nerve cord.

Identification of single-cell transcriptomes representing ITP amidated (ITPa)-expressing efferent neurons in the abdominal ganglion (iag) and other ITP neurons (non-iag) in the ventral nerve cord dataset (Allen et al., 2020). (A) Both sets of neurons express genes required for neuropeptide processing and release (amon, svr, Pal2, Phm, and Cadps) and were identified based on the neuropeptides (ITP, CCAP, AstA) they express. Dot plots showing expression of (B) monoamine, (C) neuropeptide, and (D) neurotransmitter receptors in iag and non-iag neurons.

Figure 14.. A schematic depicting ion transport peptide (ITP) signaling pathways modulating metabolic and osmotic homeostasis in Drosophila.

Different subsets of ITP neurons in the brain have been color-coded. LN_d^ITP and 5^th-LN_v are part of the circadian clock network and regulate clock-associated behaviors and physiology. L-NSC^ITP releases ITPa into the circulation following dehydration and information regarding this internal state is likely conveyed to L-NSC^ITP by other neuromodulators. Following its release into the hemolymph, ITP amidated (ITPa) activates a membrane guanylate cyclase receptor Gyc76C on the adipocytes in the fat body, principal and stellate cells in the renal tubules, as well as other targets. These signaling pathways affect diverse behaviors and physiology to modulate metabolic and osmotic homeostasis. Dashed arrows depict pathways that remain to be clarified, solid arrows represent direct effects, and red bars represent inhibition. Created with BioRender.com.

Author response image 1.

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