Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila

Abstract

The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, we sought to uncover the genetic modifiers of flight using a measure of flies' reaction and response to an abrupt drop in a vertical flight column. We conducted a genome wide association study (GWAS) using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and identified a combination of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. We functionally validated 13 of the additive candidate genes' (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo), and introduce a novel approach to whole gene significance screens: PEGASUS_flies. Additionally, we identified ppk23, an Acid Sensing Ion Channel (ASIC) homolog, as an important hub for epistatic interactions. We propose a model that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects.

Fig 1. DGRP lines show differences in flight performance across lines.

(A) The flight performance assay measures the average landing height of flies as they fall through a flight column. Vials of flies are sent down the top chute and abruptly stop at the bottom, ejecting flies into a meter-long column. Falling flies will instinctively right themselves and fly to the periphery, doing so at different times (and therefore landing at different heights) depending on their performance ability. (B-D) Collapsed z-stacks of every 10^th frame from a high-speed video recorded from the top quarter (0.25m) of the flight column illustrate these performance differences in (B) weak, (C) intermediate, and (D) strong genotypes. (E) Sexual dimorphism exists within genotypes (deviation of red dashed regression line from y = x solid gray line), though sexes are well correlated (r = 0.75, n = 197, P < 1e-36). (F) Sexually dimorphic performances are also apparent in the distribution of mean landing heights for each male (cyan) and female (red) genotype pair (mean ± S.E.M.). Sex-genotype pairs are sorted in order of increasing male mean landing height. Performances for genotypes in B-D are indicated on the distribution with the corresponding color-coded asterisk (*) above the respective genotype position.

Fig 2. Variation in flight performance associated with several additive variants, some of which were functionally validated.

(A) An additive screen for genetic variants identified several variants that exceeded the traditional [29] DGRP (P ≤ 1e-5) threshold (gray line). Significant variants (red) were spread throughout the genome on all but chromosome 4. Variants for the sex-averaged phenotype are pictured, though other sex-based phenotypes had similar profiles (S5 Fig). (B) Approximately half of all variants were shared with at least one other sex-based analysis, while the other half of all variants was exclusive to a single analysis. (C) Candidate genes were selected from the Bonferroni-corrected variants and those most significant in the sex-average analysis, for which transgenic flies were publicly available. Both sexes were tested for flight performance. Validated genes were determined if there was a significant difference between experimental lines homozygous for an insertional mutant in the candidate gene and their background control lines lacking the insertional mutant (red points, Mann-Whitney-U test, P ≤ 0.05). Very significant candidate genes (CadN, flapper (CG11073), and Dscam4) each had two independent validation lines.

Fig 3. PEGASUS_flies identifies different genetic modifiers than the additive screen.

(A) PEGASUS_flies results plotted as a Manhattan plot. For the sex-average phenotype, several genes (red points, labeled with gene symbol) exceeded a strict Bonferroni significance threshold (gray dashed line, P ≤ 3.43e-6) identified several genes. (B) PEGASUS_flies prioritizes genetic modifiers of moderate effect, taking into account linkage blocks and gene length. Significant PEGASUS_flies (red) compared against genes significant under a minSNP approach for additive variants (blue) have very little overlap between the two sets (purple). (C) Many of the genes PEGASUS_flies identified were unique to a sex-based phenotype, though the sex-average genes were generally found in other analyses.

Fig 4. Flight performance is a larger complex trait comprised of several smaller traits.

(A) The genetic architecture of epistatically interacting genes shared ppk23 as a more central node. (B) Whole genes and minSNP genes were not identified in more than three analyses, while roughly half or more genes were unique to each analysis. (C) Flight performance has a complex genetic architecture, with the key developmental gene Egfr and BMP signaling pathway contributing to wing and neurodevelopment. These processes are both important for structuring the sensory organs that enable the fly to use mechanosensory channels for proprioception. Signals from the sensory organs on the wing, head, and body travel to the brain and thoracic ganglion, which sends signals through the motor neurons to the direct and indirect flight musculature that is also differentially assembled and innervated to generate power and control the wing angle during flight.