The effect of neurospecific knockdown of candidate genes for locomotor behavior and sound production in Drosophila melanogaster

Abstract

Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination.

Keywords: BDSC; Bloomington Drosophila Stock Center; CNS; Canton-S strain; FITC; Drosophila; P element insertion; Ponce de Leon P element construct; RNAi; RNA interference; RNA interference; shRNA; Transgenic RNAi Project; UAS; Vienna Drosophila RNAi Center; candidate genes; central nervous system; CPG; central pattern generator; CS; central pattern generators, courtship song; fluorescein isothiocyanate; Gal4; green fluorescent protein; IPI; interpulse interval; LA; locomotion; locomotor activity; PBS; phosphate-buffered saline; PCR; polymerase chain reaction; PdL; small hairpin RNA; TRiP; upstream activation sequence; VDRC; yeast transcription activator protein Gal4; GFP.

Figure 1.

Locomotion parameters in flies with tissue-specific knockdown of the candidate genes (VDRC RNAi lines). GAL4/UAS-RNAi flies with spatially restricted knockdown (second column for each driver) derived by crossing elav/nrv2/appl/tsh-GAL4 drivers with VDRC RNAi lines. GAL4 controls without UAS-RNAi transgene (first, hatched columns) originate from cross of the same GAL4 drivers with host strain #60100 and are specific for each GAL4 driver. Also presented are locomotion parameters in UAS-RNAi controls descended from cross of CS_BDSC with VDRC RNAi line for a gene indicated (CS, second columns) and in flies derived by crossing CS_BDSC with host strain #60100 (CS, first, hatched column). Mean values with standard errors are shown. N = 40 for each data point. Significant difference from a corresponding control is indicated by filling (2-sided randomization test, 10,000 iterations, P < 0.05). Comparisons excluded from consideration (see text) are marked with asterisk. For details of genotypes see Methods and Table 2.

Figure 2.

Locomotion parameters in flies with tissue-specific knockdown of the candidate genes (TRiP RNAi lines). Flies with spatially restricted knockdown derived by crossing elav/nrv2/appl/tsh-GAL4 drivers with TRiP RNAi lines. The host strain #36303 was used for all control crosses. Absence of a value indicates lethality or gross morphological effects of the knockdown. Other explanations, see legend for Figure 1.

Figure 3.

Courtship song parameters in flies with tissue-specific knockdown of the candidate genes. Flies with tissue-specific knockdown derived by crossing elav/nrv2/appl/tsh-GAL4 with VDRC/TRiP RNAi lines. N = 20 for each data point. Explanations are presented in legends for Figures 1 and 2.

Figure 4.

Expression level of Sps2, CG15630 and Mef2 genes in RNAi-mediated knockdowns relative to control. Mean expression ratios (knockdown/control) with standard errors are shown. Expression levels of all candidate genes are reduced in all experimental samples (2-sided randomization test, p < 0.05, REST 2009 software).

Figure 5.

Motor parameters in flies with knockdown of CG15630 under the glia-specific repo-Gal4 driver. Glia-specific knockdown flies derived by crossing repo-GAL4 driver with VDRC RNAi lines #107797 for CG15630 and #105268 for Sps2. Control flies derived by crossing repo-GAL4 driver with host line #60100. Mean values and standard errors are shown. N = 40 for locomotor parameters, N = 20 for courtship song parameters. Significant difference from a corresponding control is indicated by filled columns (2-sided randomization test, 10,000 iterations, p < 0.05).

Figure 6.

Motor parameters in flies with estrogen-induced knockdown of CG15630 in adults. Estrogen-induced knockdown flies derived by crossing GAL4.ER driver with VDRC RNAi line #107797 for CG15630. Control flies derived by crossing GAL4.ER driver with host line #60100. Both experimental and control flies were kept on estrogen-containing medium from the moment of imago eclosion. Mean values and standard errors are shown. N = 40 for locomotor parameters, N = 20 for courtship song parameters. Significant differences between RNAi and control data are marked with asterisks (2-sided randomization test, 10,000 iterations, P < 0.05).