Modulation of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin

Abstract

Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, we delineated LK circuitry that governs various aspects of post-feeding physiology and behavior. We found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, we sought to define the populations of LK neurons that contribute to the different aspects of this physiology. We find that the calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of Lkr expression, mined a brain single-cell transcriptome dataset for genes coexpressed with Lkr, and identified synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons have different targets, but modulate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology.

Fig 1. Generation of Lk and Lkr GAL4 knock-in mutants.

(A) Schematics of the Lk and Lkr gene loci and the locations of construct insertion to generate GAL4 knock-in mutants. Note that CG34039 and ncRNA represent predictions for the presence of coding and non-coding genes in the same chromosome and overlapping location as Lk. However, there is no evidence that they are functional. Potentially, these two genes are encoded on the sense strand while Lk is on the anti-sense strand. (B) A schematic of the adult CNS showing the location of LK-expressing neurons [based on [7,8,10]]. LHLK, lateral horn LK neuron; SELK, subesophageal ganglion LK neuron; ABLK, abdominal LK neuron, T1 –T3, thoracic neuromeres. (C) Quantitative PCR shows a significant reduction in Lk and Lkr transcripts in Lk and Lkr homozygous mutants, respectively. (*** p < 0.001 as assessed by unpaired t test). (D) LK-immunoreactivity is completely abolished in the brain and ventral nerve cord of Lk mutants.

Fig 2. LK cell body size and peptide levels in Lkr mutants.

(A) LK-immunoreactivity in abdominal LK neurons (ABLKs) of Lkr mutant and control flies. (B) Staining intensity and (C) cell-body size of both the anterior (a) and posterior (p) ABLKs is increased in Lkr mutants compared to control flies. We separated the two cell groups here since the anterior (and larger) ABLKs are derived post-embryonically (during metamorphosis), and the posterior ones are functional already in the larva (see [22]). (D) LK-immunoreactivity in brain lateral horn LK neurons (LHLKs) of Lkr mutant and control flies. (E) The intensity of LK staining is unaltered in Lkr mutants. (**** p < 0.0001 as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test for C and *** p < 0.001 as assessed by unpaired t test for B).

Fig 3. Lk and Lkr mutants have altered stress resistance and water content.

Survival under desiccation is increased in both (A) Lk and (B) Lkr mutants. Survival under starvation is also increased in both (C) Lk and (D) Lkr mutants. Data are presented in survival curves, and the error bars represent standard error (**** p < 0.0001, as assessed by Log-rank (Mantel-Cox) test). (E) Hydrated and 9-hour-desiccated (9 h) Lk and Lkr mutant flies show increased water content compared to control flies. (** p < 0.01, *** p < 0.001, **** p < 0.0001 as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test).

Fig 4. Calcium activity of ABLKs under nutritional and osmotic stress.

(A) The calcium activity of ABLKs, as measured using CaLexA [23], is low in flies that have been starved, desiccated, or incubated on normal artificial food but increased in flies that have been rewatered (desiccated and then incubated on 1% agar). (B) The GFP intensity of ABLKs is increased in rewatered flies compared to other conditions. (C) The number of ABLKs that could be detected is higher in rewatered flies compared to other conditions. (assessed by one-way ANOVA followed by Tukey’s multiple comparisons test).

Fig 5. Lk and Lkr mutants show varying phenotypes in different feeding assays.

(A) Both the homozygous and heterozygous Lk mutants show decreased motivation to feed in proboscis extension reflex (PER) and this phenotype could be rescued in (B) the homozygous flies. (C) Targeted expression of tetanus toxin (to block synaptic transmission) in Lk neurons using Lk-GAL4 also caused a decrease in PER. (D) Interestingly, Lkr mutants show increased motivation to feed, which could be rescued to control levels by driving UAS-Lkr with Lkr-GAL4^CC9. See S1 Table for the statistics of graphs A-D. (E) Both the Lk and Lkr mutants show decreased long-term food intake as measured using the capillary feeding (CAFE) assay. Moreover, the homozygous mutants feed significantly lower than the heterozygous mutants (assessed by one-way ANOVA followed by Tukey’s multiple comparisons test). (F) Starved and fed Lk and Lkr mutants do not show any differences in short-term feeding compared to control flies as measured using a blue-dye feeding assay (assessed by one-way ANOVA). (G) Expression of tetanus toxin in Lk neurons with Lk-GAL4 also has no effect on short-term feeding.

Fig 6. Total activity and metabolic rate is lowered in individual Lk and Lkr mutants.

(A) Locomotor activity pattern of individual Lk homozygous and heterozygous mutants measured over 24 hours. (B) Total locomotor activity of Lk mutants is lowered compared to control flies. (C) Metabolic rate rhythms of individual Lk homozygous and heterozygous mutants measured over 24 hours. (D) Average metabolic rate of Lk mutants is lowered compared to control flies. (E) Locomotor activity pattern of individual Lkr homozygous and heterozygous mutants measured over 24 hours. (F) Total locomotor activity of Lkr mutants is lowered compared to control flies. (G) Metabolic rate rhythms of individual Lkr homozygous and heterozygous mutants measured over 24 hours. (H) Average metabolic rate of Lkr mutants is lowered compared to control flies. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as assessed by one-way ANOVA).

Fig 7. Lkr is expressed in the adult gut and Malpighian tubules.

Lkr-GAL4^CC9 drives GFP (pJFRC81-10xUAS-Syn21-myr::GFP-p10) expression in the adult (A) stellate cells in Malpighian tubules, (B) enteroendocrine cells in the posterior midgut, (C and D) anterior midgut, (E) hindgut, and (F) rectal pad. Muscles (F-actin filaments) in all the preparations (except B) have been stained with rhodamine-phalloidin (magenta). Note the expression of GFP in hindgut and rectal pad muscles. (G) Schematics of third instar larvae and adult fly showing the expression of Lkr. (Data from FlyAtlas.org, [28]). (H) A schematic of the adult gut and heat map showing expression of Lkr in different regions of the gut (R1 to R5) and its various cell types (VM, visceral muscle; EEC, enteroendocrine cell; EC, enterocyte; EB, enteroblast; ISC, intestinal stem cell; Ep, epithelium. Data was mined using Flygut-seq [29].

Fig 8. Lkr is expressed in identified peptidergic neurosecretory cells of the adult brain.

Lkr-GAL4^CC9 drives GFP (pJFRC81-10xUAS-Syn21-myr::GFP-p10) expression in (A) insulin-producing cells (labeled with anti-DILP2 antiserum) and (B) ion transport peptide (ITP)-producing lateral neurosecretory cells in the brain (labeled with anti-ITP antiserum; indicated by arrows). (C) Lkr-GAL4 drives GFP (UAS-mCD8;;GFP) expression in the adult (D and F) ITP-producing cells (indicated by the white boxes in panel C) and (E and F) insulin-producing cells (indicated by the white circle in panel C).

Fig 9. Lkr is coexpressed with peptidergic and glial markers.

Mining the single-cell transcriptome atlas of the Drosophila brain reveals that Lkr is coexpressed with (A) repo (glial marker; cell cluster marked G) and dimm (peptidergic cell marker; cell cluster marked P). (B) Within both the glial and peptidergic cell clusters, Lkr is coexpressed with ITP. Within the peptidergic cell cluster, (C) insulin-producing cells expressing DILP2, 3 and 5 could be identified (cluster marked IPCs), a subset of which express Lkr (D). Data was mined using Scope (http://scope.aertslab.org) [30]. In both (C) and (D), cells expressing all three genes are colored in white.

Fig 10. Anatomical and functional interactions between LK and insulin signaling.

(A) Expression of trans-Tango components [31] using Lk-GAL4 (from K. Asahina and D. Anderson) generates a presynaptic signal (labeled with anti-GFP antibody) in the subesophageal ganglion (SEG) and the lateral horn, and a postsynaptic signal (labeled with anti-HA antibody) in the SEG and pars intercerebralis. (B) Higher magnification of the SEG showing the presynaptic signals and the lack of post-synaptic signal in median neurosecretory cell bodies (indicated by an arrow). Note the presence of presynaptic signal in the mushroom bodies, which is due to the background noise from the trans-Tango components and not the Lk-GAL4. (C, E) Lkr homozygous mutants show increased DILP2 immunoreactivity in insulin-producing cells (IPCs) of the adult brain. (D, F) Both Lk and Lkr homozygous mutants show increased DILP3 immunoreactivity in IPCs of the adult brain. (*** p < 0.001, **** p < 0.0001, as assessed by one-way ANOVA followed by Tukey’s multiple comparisons test). CTCF, corrected total cell fluorescence.

Fig 11. Lkr knockdown in insulin-producing cells affects insulin expression and starvation resistance.

(A) Quantitative PCR shows no difference in DILP2 transcript levels between control flies (DILP2>Luciferase) and flies with Lkr knockdown in insulin-producing cells (IPCs) that were reared as adults on normal diet, high sugar and high protein (HSHP) diet, or low sugar and high protein (LSHP) diet. (B) DILP3 transcript levels are upregulated in DILP2>Lkr-RNAi-#2 (BL#65934) flies reared on normal and HSHP diets. (C) DILP5 transcription is downregulated in DILP2>Lkr-RNAi-#2 (BL#65934) flies reared on normal diet. (* p < 0.05 and ** p < 0.01 as assessed by unpaired t test). Flies maintained as adults on (D) normal diet and (E) HSHP diet show increased starvation resistance whereas flies maintained on (F) LSHP diet have similar survival under starvation compared to control flies. For graphs D-F, data are presented in survival curves and the error bars represent standard error (**** p < 0.0001, as assessed by Log-rank (Mantel-Cox) test).

Fig 12. Lk signaling scheme.

LK signaling scheme showing the location of all LK neurons, identified neurons downstream of LK neurons, target tissues, based on Lkr distribution and effects of LK signaling. Dashed arrows indicate probable links that need to be functionally validated. DSK, drosulfakinin; sNPF, short neuropeptide F; DTK, tachykinin.