Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits

Abstract

Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging's multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). We examined if foraging's complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, we cloned DNA fragments upstream of each of foraging's transcription start sites and generated four separate for^pr-Gal4s. Supporting our hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. In the CNS, for^pr1-Gal4 and for^pr4-Gal4 were expressed in neurons while for^pr2-Gal4 and for^pr3-Gal4 were expressed in glia cells. In the gastric system, for^pr1-Gal4 and for^pr3-Gal4 were expressed in enteroendocrine cells of the midgut while for^pr2-Gal4 was expressed in the stem cells of the midgut. for^pr3-Gal4 was expressed in the midgut enterocytes, and midgut and hindgut visceral muscle. for^pr4-Gal4's gastric system expression was restricted to the hindgut. We also found promoter specific expression in the larval fat body, salivary glands, and body muscle. The modularity of foraging's molecular structure was also apparent in the phenotypic rescues. We rescued larval path length, triglyceride levels (bordered on significance), and food intake of for⁰ null larvae using different for^pr-Gal4s to drive UAS-for^cDNA. In a foraging null genetic background, for^pr1-Gal4 was the only promoter driven Gal4 to rescue larval path length, for^pr3-Gal4 altered triglyceride levels, and for^pr4-Gal4 rescued food intake. Our results refine the spatial expression responsible for foraging's associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging's pleiotropy arises at least in part from the individual contributions of its four promoters.

Keywords: Drosophila melanogaster; foraging gene; larval phenotypes; multiple promoter analysis; pleiotropy.

Figure 1:. Cloning of for^pr-Gal4s.

A) Schematic of the foraging locus (dark grey - exons), its transcripts above the locus (grey - UTR, black - ORF), transcription start sites (arrows) and the cloned regions (colored bars below locus) used in the promoter analysis. The regions up to 5kb upstream of and 300bp downstream of the TSS for the four identified minimal promoters were cloned into a gypsy insulated Gal4 vector. The ORF used in the UAS-for^cDNA is annotated below the transcripts and is a derivative of the RNA transcript A (RA) and codes for the protein isoform A (PA). B) Example of one of the cloned for^pr1-Gal4 constructs. The for^pr1-Gal4 segment is flanked by gypsy insulator sequences. An attB site-specific recombination sequence from φC31 was added to the vector. C) PCR confirmation of the four for^pr-Gal4 integrations. There was a positive integration event in all lines.

Figure 2:. for^pr-Gal4s expression in the 3^rd instar larval CNS, gastric system, and fat body.

Whole-mount immunofluorescence analysis of for^pr-Gal4 driving UAS-mCD8::GFP in the larval CNS and stained with anti-GFP. Sample sizes were n ≥ 12 larvae for each of experiment. Representative images are shown. Scale bars = 100µm. A) Schematic of the foraging locus with the cloned regions near the transcription start sites highlighted. B) for^pr1-Gal4 expressed in neurons throughout the VNC and brain lobes. C) for^pr2-Gal4 expressed in midline glia in the VNC. D) for^pr3-Gal4 expressed in the perineurial surface glia of the CNS and PNS. E) for^pr4-Gal4 expressed in the optic lobes of the CNS and the eye imaginal discs and the leg imaginal discs. F) for^pr1-Gal4 expressed in enteroendocrine cells in the anterior portion of the larval midgut. G) for^pr2-Gal4 expressed in the adult midgut precursor cells (AMP) throughout the midgut. H) for^pr3-Gal4 expressed in the muscle of the midgut. I) for^pr4-Gal4 expressed in the h5d, h6d, hv, and h7 regions of the larval hindgut. J) for^pr1-Gal4 was not expressed in fat body. K) for^pr2-Gal4 was not expressed in fat body. L) for^pr3-Gal4 was expressed in the fat body. M) for^pr4-Gal4 was not expressed in fat body.

Figure 3:. for^p1-Gal4 rescues foraging null (for⁰ ) larval path length.

A) The pr1 experimental (for⁰/for⁰; {for^pr1-Gal4}/{UAS-for^cDNA}) had significantly longer path lengths than either the Gal4 control (for⁰/for⁰; {for^pr1-Gal4}/+, p=0.00041) and the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.0004). B) The pr2 experimental (for⁰/for⁰; {for^pr2-Gal4}/{UAS-for^cDNA}) did not have significantly longer path lengths than the Gal4 control (for⁰/for⁰; {for^pr2-Gal4}/+, p=0.95). C) The pr3 experimental (for⁰/for⁰; {for^pr3-Gal4}/{UAS-for^cDNA}) was not significantly different from the Gal4 control (for⁰/for⁰; {for^pr3-Gal4}/+, p=0.93) or the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.89). D) The pr4 experimental (for⁰/for⁰; {for^pr4-Gal4}/{UAS-for^cDNA}) was not significantly different from the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.17). Sample size: n=30 larvae per genotype per plot. The boxplots show the mean with the whiskers displaying 1.5 times the interquartile range. For a rescue to be considered significant the UAS/Gal4 treatment had to be significantly different than both of the UAS and the Gal4 controls.

Figure 4:. for^p3-Gal4 affects larval triglyceride levels.

A) The pr1 experimental (for⁰/for⁰; {for^pr1-Gal4}/{UAS-for^cDNA}) was not significantly different from the Gal4 control (for⁰/for⁰; {for^pr1-Gal4}/+, p=0.78) or the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.94). B) The pr2 experimental (for⁰/for⁰; {for^pr2-Gal4}/{UAS-for^cDNA}) was intermediate between the significantly different Gal4 (for⁰/for⁰; {for^pr2-Gal4}/+) and UAS (for⁰/for⁰; +/{UAS-for^cDNA}) controls. C) There was significant variation in the pr3 triglyceride experiment (F_(2,27)=3.76, p=0.036). The pr3 experimental (for⁰/for⁰; {for^pr3-Gal4}/{UAS-for^cDNA}) bordered on being significantly different from the Gal4 control (for⁰/for⁰; {for^pr3-Gal4}/+, p=0.06) and the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.06). D) The pr4 experimental (for⁰/for⁰; {for^pr4-Gal4}/{UAS-for^cDNA}) was not significantly different from the Gal4 control (for⁰/for⁰; {for^pr4-Gal4}/+, p=1.00). Sample size: n=10 extracts (of 10 pooled larvae) per genotype per plot. The boxplots show the mean with the whiskers displaying 1.5 times the interquartile range. For a rescue to be considered significant the UAS/Gal4 treatment had to be significantly different than both of the UAS and the Gal4 controls.

Figure 5:. for^p4-Gal4 rescues null larval food intake.

A) The pr1 experimental (for⁰/for⁰; {for^pr1-Gal4}/{UAS-for^cDNA}) was not significantly different from the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.85). B) The pr2 experimental (for⁰/for⁰; {for^pr2-Gal4}/{UAS-for^cDNA}) was not significantly different from the Gal4 control (for⁰/for⁰; {for^pr2-Gal4}/+, p=0.87) or the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.86). C) The pr3 experimental (for⁰/for⁰; {for^pr3-Gal4}/{UAS-for^cDNA}) was not significantly different from the Gal4 control (for⁰/for⁰; {for^pr3-Gal4}/+, p=0.18). D) The pr4 experimental (for⁰/for⁰; {for^pr4-Gal4}/{UAS-for^cDNA}) was significantly different from the Gal4 control (for⁰/for⁰; {for^pr4-Gal4}/+, p=0.00005) and the UAS control (for⁰/for⁰; +/{UAS-for^cDNA}, p=0.006). Sample size: n=10 extracts (of 10 pooled larvae) per genotype per plot. The boxplots show the mean with the whiskers displaying 1.5 times the interquartile range. For a rescue to be considered significant the UAS/Gal4 treatment had to be significantly different than both of the UAS and the Gal4 controls.

Figure 6:. Summary of expression and phenotypic effects.

A schematic of the foraging gene in the center with exons in grey and transcription start sites marked with arrows. The regions cloned in the for^pr-Gal4s are colored and shaded. The expression patterns seen in the for^pr-Gal4s are summarized above the locus. Depictions of the rescued phenotypes are shown below the locus.