Figures and data
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. Grey 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 Fig. S1A) 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-NSCITP), one pair of diuretic hormone 31 (DH31)-expressing lateral neurosecretory cells (L-NSCDH31), one pair of 5th ventrolateral neurons (5th-LNv) and one pair of dorsolateral neurons (LNdITP). L-NSCITP and L-NSCDH31 are a subset of lateral neuroendocrine cells and the single pairs of 5th-LNv and LNdITP 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 1 Supplement 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-NSCITP, 5th-LNv and LNdITP 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. Grey box indicates presence and white box indicates absence.
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 ITP 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.
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 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 labelled by Period antibody and marked by arrowheads). Gyc76C is not expressed in (N) insulin-producing cells (labelled by DILP2 antibody) and (O) adipokinetic hormone (AKH) producing endocrine cells but is expressed in the corpora allata (CA) (marked in white).
Recombinant Drosophila ITPa inhibits Malpighian tubule secretion via Gyc76C.
(A) Schematic of Ramsay assay used to monitor ex vivo secretion by tubules. (B) Application of Drosophila 500nM ITPa does not affect basal secretion rates by unstimulated tubules. 500nM ITPa inhibits both (C) 10nM leucokinin (LK)-stimulated and (D) 1μM diuretic hormone 31 (DH31)-stimulated secretion rates. Importantly, while 500nM ITPa inhibits (E) 10nM LK-stimulated secretion and (F) 1uM 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. For B-D, * p < 0.05 and **** p < 0.0001 as assessed by unpaired t test. For E and F, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as assessed by two-way ANOVA followed by Šidák’s multiple comparisons test.
ITPa is released during desiccation to impact osmotic and ionic stresses.
ITPa immunofluorescence, indicative of peptide levels, is lowered in 5th-LNv, LNdITP and L-NSCITP of (A) male and (B) female flies exposed to desiccation. ITPa peptide levels recover to control levels in flies that were rehydrated following desiccation. Lower peptide levels during desiccation indicates increased release. (C) Adult-specific overexpression of ITPa using ITP-RC-GAL4TS (ITP-RC-T2A-GAL4 combined with temperature-sensitive tubulin-GAL80) increases desiccation. (D) ITPa overexpression also results in increased water content and (E) a slightly bloated abdomen (marked by an asterisk). Knockdown of Gyc76C with UAS-Gyc76C RNAi (#106525) in both the (F) principal cells of renal tubules using uro-GAL4 and (G) stellate cells using c724-GAL4 reduces desiccation tolerance. Gyc76C knockdown in (H) principal cells increases survival under salt stress whereas knockdown in (I) stellate cells lowers survival. (J and K) Gyc76C knockdown in principal or stellate cells increases the time taken for recovery from chill-coma. For A, B and D, * 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 C, F-K, ** p < 0.01, **** p < 0.0001, as assessed by Log-rank (Mantel-Cox) test.
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 hours), (G) a preference for yeast over sucrose, and (H and I) defects in preference for nutritive sugars when starved for 4 hours prior to testing. Flies with Gyc76C knockdown in the fat body have (J) diminished ovaries, they (K) defecate more and have (L) reduced water content than the controls. For K, number of excreta counted over 2 hours. Gyc76C knockdown also impacts (M) DILP2 peptide levels (N) but not 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 night-time 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.
Adult-specific ITPa overexpression using ITP-RC-T2A-GAL4 impacts metabolic homeostasis, feeding and related behaviors.
Overexpression of ITPa using ITP-RC-GAL4TS (ITP-RC-T2A-GAL4 combined with temperature-sensitive tubulin-GAL80) (A) increases starvation tolerance. ITPa overexpression results in (B) reduced circulating glucose levels but has no effect on (C) glycogen levels. (D) The size of neutral lipid droplets (stained with Nile red) is increased in flies with ITPa overexpression. (E and F) These flies also exhibit defects in preference for nutritive sugars when starved for 16 hours prior to testing. (G) ITPa overexpression flies have enlarged ovaries. (H) ITPa overexpression has no effect on locomotor activity under fed or desiccating conditions. All experiments were performed at 29°C. For A, **** p < 0.0001, as assessed by Log-rank (Mantel-Cox) 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.
Inputs and outputs of ITP neurons based on connectomics and single-cell transcriptomics.
(A) Reconstruction of 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-NSCITP) are shown in grey, whereas the 5th ventrolateral neurons (5th-LNv) and dorsolateral neurons (LNdITP) are shown in black. Diuretic hormone 31 (DH31)-expressing lateral neurosecretory cell (L-NSCDH31) is not shown since it is not clear which of the three L-NSCDH31 co-expresses ITPa. Site of input synapses are marked in magenta and output synapses are marked in green. L-NSCITP do not receive any significant synaptic inputs within this brain volume and were thus excluded from subsequent analyses. (B) Number of neurons (categorized by neuronal super classes annotated in the FlyWire connectome (Schlegel et al., 2023)) providing inputs to and receiving outputs from 5th-LNv and LNdITP. Reconstructions of neurons from different super classes (C) providing inputs to and (D) receiving outputs from 5th-LNv and LNdITP. Central neurons are multi-colored for clarity. (E) Identification of single-cell transcriptomes representing different subsets of ITPa-expressing neurons in the adult brain dataset (Davie et al., 2018). Since both the 5th-LNv and LNdITP co-express ITP, cryptochrome (cry) and neuropeptide F (NPF), these cells are grouped as LNITP. 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 (F) monoamine, (G) neuropeptide and (H) neurotransmitter receptors in different sets of ITPa neurons.
A schematic depicting ITP signaling pathways modulating metabolic and osmotic homeostasis in Drosophila.
Different subsets of ITP neurons in the brain have been color-coded. LNdITP and 5th-LNv are part of the circadian clock network and regulate clock-associated behaviors and physiology. L-NSCITP release ITPa into the circulation following dehydration and information regarding this internal state is likely conveyed to L-NSCITP by other neuromodulators. Following its release in the hemolymph, 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 indicate indirect inputs, solid arrows represent direct effects, and red bars represent inhibition. Created with BioRender.com.
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