Chapter V: Discussion and future directions
5. Other potential neuromodulators mediating the effects of
5.3 DILP-7
In Drosophila, there are seven genes encoding DILPs. The DILPs I have been referring to throughout my dissertation are DILP-2, DILP-3, and DILP-5, which are expressed in the pars intercerebralis and have functional roles in growth and metabolism [5]. DILP-6 is expressed in the fat body and also plays a role in growth and development, as it is required to maintain growth during the non-feeding stage of metamorphosis [81, 82]. In contrast, DILP-7 is not essential for growth and development, but instead is involved in food-based decision-making.
The evidence against the involvement of DILP-7 in growth and development is that flies with a null mutation in DILP-7 exhibit normal development time, body weight, lipid and glycogen content, starvation and paraquat resistance, viability, lifetime fecundity, or median lifespan [83]. Further support for this suggestion, is that both the survival and body weight of flies with combinatorial mutations in DILP-2, -3, and -5 or in DILP-1–5 were significantly reduced, and combining these combinatorial mutants with a null mutation in DILP-7 did not further decrease survival or body weight. While not being required for growth and development, and not being sufficient to compensate for the loss of other DILPs, an independent study demonstrated that constitutive overexpression of DILP-7 using armadillo-Gal4 (a housekeeping gene, thus expressed everywhere) resulted in a increase in body weight of adults [20]. This effect on body weight was likely due to overexpressing DILP-7 at much higher levels throughout the body than might be expected from the endogenous DILP-7 enhancer. Evidence for low levels of DILP-7 endogenous expression levels is that DILP-7 expression was not detectable by in situ hybridization in adults [20].
Several studies have implicated DILP-7 in the regulation of feeding behavior. One study demonstrated a role for DILP-7 neurons in the rate of food intake. Silencing DILP-7 neurons did not have any effect on food intake unless provided a low-nutrient food source, in which case food intake was increased compared to control flies [24]. Feeding behavior was increased during 24 and 48 hours of feeding on 26 mM sucrose plus 0.6% yeast, whereas food intake was normal when provided a nutritious diet, which contained sixfold the amount of sucrose and yeast.
Another study demonstrated a role for DILP-7 in determining oviposition site.
Overexpression of DILP-7 by either using hs-DILP-7 (a heat shock promoter fused to DILP-7) or by using DILP-7-Gal4/UAS-DILP-7 reduced or eliminated the oviposition preference of females, respectively [84]. Whereas wild-type females preferentially laid eggs on lobeline when given the choice between this aversive tastant and sucrose, over- expressing DILP-7 either suppressed or eliminated this preference. The nature of this oviposition preference of wild-type flies is peculiar, given the stimulatory value of the sucrose (100 mM) and the lack of nutritional value of the lobeline (which did not contain any sucrose). Both oviposition sites contained 1% ethanol, which might have added stimulatory value to the lobeline. The effects of overexpressing DILP-7 were not further examined in this study.
In wild-type flies, nutritional state did not effect the decision to lay eggs on lobeline, and wild-type flies visited either food source an equal number of times [84]. This suggests that flies do not indiscriminately choose the location to lay eggs or that nutritional state would alter this choice. In fact, the preference against laying eggs on 100 mM sucrose of wild-type females was upheld when given the choice between sucrose and agarose. These findings suggest that overexpressing DILP-7 is not reflecting the behavior of a “sated” fly. When DILP-7 neurons were silenced, using DILP-7-Gal4/UAS-Kir2.1, all egg-laying motor patterns were abolished, rendering these flies sterile. In addition to the expression pattern of DILP-7 in both the female reproductive tract and the SOG, this suggests that DILP-7 signaling may be involved in integrating gustatory cues to inititate oviposition motor patterns.
The expression pattern of AstA and DILP-7 in the larval brain is surprisingly similar (data not shown). Similar to AstA expression in the VNC, DILP-7 is expressed in several pairs of large abdominal ganglion neurons that project to the hindgut and rectum, in three pairs of lateral dorsal neurons, and in a pair of central medial neurons [84].
Immunostaining images of DILP-7 in the adult brain in Yang et al. [84], shows a strong signal in the posterior SOG, in the same location that AstA and AstA-Gal4 label a pair of cell bodies. In contrast to antibody staining, DILP-7-Gal4 was not expressed in the dorsal medial SOG, which could be explained if additional enhancers of DILP-7 expression were missing in the DILP-7-Gal4 transgenic construct. It is also possible that the DILP-7 antibody is not specific, and is detecting the expression of other DILPs in the SOG. In the
adult, the DILP-7 cell bodies in the abdominal ganglion send projections through cervical connective to the SOG and also to the hindgut, rectum, and to the female reproductive tract.
Although the AstA-Gal4 transgenics and the anti-AstA antibody do not show expression in the female reproductive tract, it is possible that the VNC neurons that project to the hindgut and rectum overlap with DILP-7 neurons. Both the AstA-Gal4 transgenics and the anti- AstA antibody also show expression in the ventral nerve cord and the SOG, but the origin of the projections in the cervical connective is not clear.
This similarity in expression patterns is especially intriguing due to the proximity of Drosophila AstA receptor 1 (DAR-1) and Drosophila insulin-like peptide 7 (DILP-7) on the X chromosome (DAR-1 is located only ~ 1.3 kb upstream of DILP-7 and in the same 5’
to 3’ orientation). The close proximity of two genes involved in feeding decision-making suggests that there may be common enhancers for these genes. Although microarray data suggest that DAR-1 is exclusively expressed in the CNS [40], the expression pattern of DAR-1 is undetermined because in situ hybridization of GPCRs has generally posed a challenge.
These observations suggest a possible overlap between DILP-7 and either AstA or DAR-1 expression. Arguing against DILP-7 and AstA co-expression though, is a report that larval DILP-7 neurons weakly express proctolin, which is a widely expressed neuropeptide that stimulates gut contractions. In several insects, AstA has been reported to inhibit proctolin-induced gut contractions [8, 85]. But since cases/examples exist where neuropeptides with counteracting functions are co-expressed, this finding may not exclude the possibility that AstA and DILP-7 are co-expressed.
Also intriguing, is the fact that DILP-7 is the most conserved DILP in Drosophila, and is in fact the only DILP with homology to other ILPs outside of Drosophila species.
Similarly, AstA is found in almost all insects/arthropods studied, and at least 431 different isoforms of AstA have been observed in arthropods (!!). The extent of conservation of each of these molecules suggests an important role in evolution. Although the sequence of the neuropeptide AstA has not been conserved across the animal kingdom, the sequence of the receptors of AstA, DAR-1 and DAR-2, has been conserved. In C. elegans and in mammals, receptor orthologues of DAR have similarly been implicated in regulating feeding behavior. The close proximity of DILP-7 and DAR-1 on the X chromosome may
have co-conspired in the evolutionary conservation of these two signaling systems.
Evidence for a role of DILP-7 in food-based decision-making without displaying a role in growth and development, in combination with potential overlap of DILP and AstA/DAR1 expression, begs the question of whether DILP-7 signaling is driving the phenotype we observed upon AstA activation, an impairment of feeding decision-making without a role in metabolism. Another possibility is that these two signaling systems are interacting. I can address this possibility by using the feeding and oviposition assays described in the DILP-7 studies to examine whether AstA/Kir2.1 flies exhibit similar phenotypes as DILP-7/Kir2.1 flies. Further characterization of DILP-7 overexpression is necessary as well as characterization of DILP, AstA, DAR-1, and InR expression patterns.
Given the number of DILP-7 tools available, we could also determine whether AstA- Gal4/UAS-DILP phenocopies the observed phenotype of activating AstA neurons, and whether AstA-Gal4/UAS-NaChBac/UAS-DILP-7-RNAi rescues the phenotype.