In Drosophila melanogaster, two members of the Ski/Sno protein family exist, the Ski novel oncogene (Snoo) (Barrio et al. 2007) and the functional Smad suppressing element (Fuss) (Fischer et al. 2012). We have previously shown that Fuss is specifically expressed in gustatory receptor neurons (GRNs) and interneurons in the central nervous system (CNS). We created a loss of function allele fussdelDS and showed that loss of fuss had no impact on survival rate during development or on adult life span. In fact, fussdelDS flies display an impaired bitter GRN development and bitter taste reception (Rass et al. 2019).
To address if fussdelDS shows other behavioural phenotypes we tested these flies in different behavioural assays (circadian rhythm, optomotor response, climbing and Buridan’s paradigm), but we only found phenotypes in a climbing (Botella et al. 2004) and Buridan’s paradigm assay (Bülthoff et al. 1982; Colomb et al. 2012). In the climbing assay, flies were allowed to climb as high as possible in a pipette within 12 seconds. Interestingly, although fuss is not expressed in motoneurons, a strong reduction of the mean climbing speed of homozygous mutant fussdelDS flies was detected if compared to heterozygous controls (fussdelDS /+) (Fig 1, A). Upon closer examination of this phenotype, we noticed that the flies did not show any impairment in climbing capability but rather exhibited an increased number of pauses during testing. To record several different traits, we chose to test the flies using the Buridan’s paradigm assay. One day old flies’ wings were clipped, flies were allowed to recover for one night and underwent testing the next day. Flies were placed on a platform surrounded by water. The arena is homogenously illuminated except for two black stripes on opposite sides. If flies perceive the stripes, they will try to reach them mainly in an alternating fashion. The movement of every fly was recorded for 10 minutes with BuriTrack and analysed with the CeTrAn analysis software (Colomb et al. 2012). Interestingly, no difference between mutant and control flies in speed could be observed anymore, because the speed in this assay is only measured during activity (Fig 1, B). Furthermore, the number of walks between stripes was not significantly different between genotypes and showed only a slight tendency to a lesser number of walks between stripes in mutant flies (Fig 1, C). However, the overall activity time of mutant flies was significantly reduced in contrast to control flies (Fig 1, D). Similar to our previous observation, the number of pauses was strongly increased in mutant flies (Fig 1, E), whereas the pause length was unchanged (Fig 1, F). Overall, we can say that, due to the increased number of parameters recorded via BuriTrack and analysed with CeTrAn, the reduced climbing speed observed in the climbing assay is not the consequence of slower walking, but rather due to an increased number of pauses during walking, which ultimately leads to a reduced locomotor activity. Additionally, we have found that mutant fussdelDS flies have no problems in perceiving the black stripes as shown by the average trajectory plot and the stripe deviation (Fig 1, G).
In future experiments, it will be of particular interest how the loss of fuss impairs the interneurons on a molecular and cellular level and if these interneurons are somehow synaptically connected to motoneurons, which would explain the increased number of walking pauses. In humans a reduced expression of the fuss homolog Skor1 has been shown to be linked to Restless Legs Syndrome (RLS) (Catoire et al. 2018). However, it is rather unlikely that fly fuss has a similar function as human Skor1, or that fuss mutant flies could serve as a model for RLS, because a loss of function dBTBD9 RLS fly model is associated with a higher activity and a reduced number of pauses in Buridan’s assay (Freeman et al. 2012).
MethodsRequest a detailed protocol
Generation and characterisation of the fussdelDS (FBal0349586) allele has been described before (Rass et al. 2019). The fussdelDS allele has been introduced into wild-type Berlin (WTB) background by backcrossing it five times to WTB flies. In the behavioural assays, only male flies underwent testing and homozygous fussdelDS flies were crossed to WTB and heterozygous progeny served as controls. fussdelDS/+ control flies did not show differences compared to WTB males. In climbing and Buridan´s assays, thirteen control flies and fifteen mutant flies have been analysed. For Buridan´s assay, we used the devices and software described previously in Colomb et al. (2012). For data visualisation the Python libraries pandas, matplotlib and seaborn have been used. For statistical testing the Python library SciPy was used.
This work was supported by the Deutsche Forschungsgemeinschaft (FI2133/1-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Reviewed BySteven Marygold and Anonymous
HistoryReceived: December 19, 2019
Accepted: March 6, 2020
Published: March 9, 2020