Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides

Abstract

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.

Figure 1. Schematic depiction of a subset of peptidergic lateral neurosecretory cells in the adult Drosophila brain.

The ipc-1 and ipc-2a cells have large cell bodies and co-express the peptides ITP, DTK and sNPF, as well as the enhancer trap Gal4 lines c929 (transcription factor DIMM) and Kurs-6. These neurosecretory cells have axons extending through the corpora cardiaca nerves (NCC) to varicose terminations in the corpora cardiaca, anterior aorta and anterior intestine. Another set of neurons (LNC) in the same cluster as ipc-2a express either sNPF or DTK, but not ITP, c929 or Kurs-6. Their axon trajectories are not known.

Figure 2. A set of ten large neurosecretory cells coexpress ITP and the transcription factor DIMM.

Coexpression of c929-driven GFP (Green; representing transcription factor DIMM) and immunolabeling with antiserum to ITP (magenta) in a subset of neurosecretory cells (ipc-1 and 2a) of the adult Drosophila brain. The images are from frontal views of wholemount specimens (dorsal is up). Ai - iii Overview of dorsal brain with ipc-1 (asterisks) and 2a neurons with coexpression of ITP and c929 (stack of several confocal sections). Note that cell bodies of the ipc-1 neurons have variable positions (along the same axonal tract; see also Fig. S1). Aiv Enlarged view of area that is boxed in Aiii. Note that there are three more ITP-labeled ipc-2 neurons (two seen at asterisks; the third is in adjacent optical section) that do not express c929. Bi - iii Higher magnification of the same section with colocalized markers in ipc-1 and 2a cells. Only one ipc-1 is seen to the left in this optical section and four to the right. The asterisks indicate two of the small ipc-2 cells that do not express c929-GFP. A set of presumed lateral neurosecretory cells (LNC) express c929, but not ITP.

Figure 3. Co-expression of peptide and c929 or Kurs-6 Gal4 expression in neurosecretory cells of the adult brain.

All images show brain in frontal view; Gal4-GFP expression is shown in green immunolabeling in magenta. Ai - iii Co-expression of DTK-immunolabeling and c929 expression in ipc-1 and 2a cells. These neurosecretory cells are the only ones coexpressing the two markers. Bi - iii Co-expression of ITP immunolabeling and snpf-GAL4 –driven GFP in ipc-1 and 2a cells. Note the variable location of ipc-1 cell bodies. The intrinsic Kenyon cells (KC) of the mushroom bodies express sNPF and their dendrites in the calyces (Ca) are seen here. Ci - iii The same section in higher magnification showing double labeled ipc-1 cell bodies in the right brain hemisphere. In Cii the ipc-1 cells are indicated by asterisks. Di - iii Co-expression of ITP-immunolabeling and Kurs-6-Gal4 expression in ipc-1 and 2a cells. Note that only one (the large ipc-2a) of the four ipc-2 cells coexpress the two markers.

Figure 4. sNPF-immunolabeling in axons supplying the retrocerebral complex.

Wholemount preparations of the foregut (or esophagus), proventriculus (prov) with attached corpora cardiaca-recurrent nerve and corpora cardiaca-hypocerebral ganglion (corpora allata not seen here). As a marker to outline the retrocerebral complex Dilp2-Gal4-driven GFP was used to reveal axons of insulin producing cells. It is clear that the DILP2 expressing median neurosecretory cells in the brain do not co-express sNPF or DTK (not shown). Thus, the occational “white profiles” in the merged images are caused by close superposition of two separate neurons. Ai - iii Foregut and proventriculus with the corpora cardiaca nerve (arrow) and corpora cardiaca with hypocerebral ganglion (boxed) with axons expressing Dilp2 and sNPF-immunolabel. These panels show projections of several optic sections, thus some Dilp2 and sNPF expressing axons superimpose in Aiii. B A higher magnification of the corpora cardiaca nerve separate varicose axons express Dilp2 and sNPF (no colocalization). Ci - iii Detail of the corpora cardiaca region with the two markers in separate varicose axons.

Figure 5. In the brain of the third instar larva the large neurosecretory cells do not coexpress marker.

The brain of later third instar larva is shown in dorsal view, anterior is at top of images. The Gal4-GFP expression is shown in green, immunolabeling in magenta. Ai – iii The four pairs of ITP immunolabeled ipc-1 cells do not express c929 (single channels are shown in ii and iii). Bi – iii Only two neurons (arrows) coexpress DTK immunolabeling and c929 (single channels in ii and iii). These are descending interneurons with extensive axonal projections into the ventral nerve cord (not shown). C The ipc-1 neurons coexpress ITP immunolabel and Kurs 6 expression (this GFP is seen also in nuclei). D The four pairs of ipc-1 neurons express Kurs 6, but not DTK immunolabel. The large descending neurons (arrows), however, coexpress the two markers.

Figure 6. Knockdown of sNPF and DTK in neurosecretory cells increases sensitivity to desiccation.

Two different Gal4 drivers that specify the ipc-1 and 2a cells were used to knock down levels of DTK or sNPF in these cells. Flies were kept singly in tubes with neither food nor water and each hour (starting after 12 h) dead flies were counted. Each of the four knockdown experiments (red curves) displayed a significant decrease in survival at desiccation. As controls the two parental strains crossed to w¹¹¹⁸ flies were used. All experiments were run in triplicate (three separate fly crosses per genotype) with a minimum of 40 flies of each genotype in each replicate (except for one control in B and D) The statistics used for trends in survival was a Log-rank test (Mantel-Cox). The survival curves are displayed as averages of the three replicates with standard errors indicated by bars. A Knockdown of DTK in ipc-1 and 2a cells with the c929 enhancer trap Gal4 line (C929) crossed with UAS-Dtk-RNAi (DtkRi). The survival curve of knockdown flies was significantly different from that of the two controls (P<0.001, Log-rank test; for each genotype n = 139–171). B Flies bearing the transgenes c929-Gal4 and UAS-snpf-RNAi (sNPFRi) displayed a similar decrease in survival at desiccation [P<0.001, Log-rank test; for each genotype n = 153, 154, 96 (sNPFRi x W¹¹¹⁸)]. C The Kurs 6 Gal4 driver produced a drastic phenotype when crossed to UAS-Dtk-RNAi flies. A dramatic decrease in survival was observed: median lifespan of peptide knockdown flies was reduced by 10 h compared to the controls (P<0.001, Log-rank test; for each genotype n = 120). D The flies from the cross between Kurs 6 and UAS-snpf-RNAi displayed a drastic reduction in survival [P<0.001, Log-rank test; for each genotype n = 146, 178 and 96 (sNPFRi x W¹¹¹⁸)].

Figure 7. Water loss increases in peptide-knockdown flies exposed to desiccation.

Flies with DTK diminished with the Kurs 6 Gal4 driver were exposed to desiccation (no food and no water) for 16 h. The whole body water content was calculated in the desiccated flies and in flies of the same genotypes that were normally fed (see Material and methods). The water loss is given in the graph for the experimental and control flies. Each genotype was tested in three replicates. A significant increase in water loss was seen in the flies with diminished DTK in ipc-1 and 2a cells (One-way ANOVA, with Bonferroni's multiple comparison, P<0. 001; for each genotype n = 100–170).

Figure 8. Sensitivity to starvation increases after knockdown of sNPF and DTK in neurosecretory cells.

The two Gal4 drivers, c929 and Kurs 6 were used to specify knock down levels of DTK or sNPF in the ipc-1 and 2a cells. Flies were kept individually in tubes supplied with aqueous agarose, but no food. Dead flies were monitored every 12 h and experiments were run in triplicates (n =  at least 46 flies per genotype and replicate), otherwise the experimental procedure and statistics were as in Fig. 6. In each of the experiments the peptide knockdown resulted in flies with significantly diminished survival at starvation. A Knockdown of DTK in ipc-1 and 2 cells with the c929 driver (P<0.001, Log-rank test; for each genotype n = 139–171). B Knockdown of sNPF with c929 driver (P<0.001, Log-rank test; for each genotype n = 166–247). C Kurs 6-driven knockdown of DTK produces strongly diminished survival at starvation (about 12 h decrease at 50% survival) (P<0.001, Log-rank test; for each genotype n = 224–229).

Figure 9. Locomotor activity in transgenic flies at starvation.

Flies were tested for locomotor activity during starvation. DTK knockdown flies generated by the cross of Kurs 6-Gal4 and UAS-Dtk-RNAi flies (Kurs 6-DtkRi) and parental controls (w¹¹¹⁸-Kurs 6 and w¹¹¹⁸-DtkRi) were kept individually in glass tubes with aqueous agarose in one end, under 12∶12 light:dark (LD) conditions at 25°C, and their locomotor activity was recorded in a Trikinetics activity monitor system. We display average activity (arbitrary units) of all flies of each genotype over 40 h, starting 3 h after onset of starvation. In the graph we set the time 0 h at onset of recording (which started 5.5 h after lights on). The LD phases are indicated by yellow/black bar below. Flies of all three genotypes displayed the same locomotor activity patterns until 18 h of recording. At the end of the light phase an increased activity was seen, corresponding to evening activity (evening anticipation). This declines over about 2 h into the dark phase where a trough level is reached at about 8 h of monitoring. The flies thereafter increase their activity to a steady intermediate level over the remaining recording. This increased activity during the dark phase is likely to reflect food search activity. At 18 h the peptide knockdown flies start to die, but continued recording shows that control flies continue the same level of activity and no morning or evening peak of activity can be seen on day two. The experiment was made in two replicates with a total of 56–81 flies of each genotype still alive at 18 h of recording.