Article Figures & Data
Figures
- Figure 1.
Shal is expressed in JO neurons and localizes to sensory dendrites and somata. A, Immunostaining of pupal JO from the Shal protein trap line, ShalMI00446-GFSTF.1. The tagged Shal protein is stained with anti-EGFP and anti-FLAG in the green channel. Neurons are visualized with anti-HRP (magenta) which shows enhanced dendrite staining. Phalloidin (blue) stains the scolopale rods in the scolopale cell surrounding the sensory dendrite. Brackets indicate the inner (white bracket) and outer (yellow bracket) dendritic segments. White-dotted outline indicates an example cilium. Scale bar, 5 μm. B, Immunostaining of pupal JO from flies expressing the Shal dominant-negative construct, UAS-HA-ShalW362F, in neurons. Anti-HA staining shows the dominant-negative construct in green, with anti-HRP (magenta) and phalloidin (blue). Brackets and white-dotted outline as in A. Scale bar, 5 μm. For expression of Shal in the Fly Cell Atlas antennal single nucleus RNA sequencing data, see Extended Data Figure 1-1.
- Figure 2.
Shal loss of function impairs auditory signals in the antennal nerve. A, Schematic of electrophysiological recording preparation. SEPs are recorded as differential field potentials from a tungsten electrode placed near the antennal nerve (recording electrode) relative to one inserted in the dorsal head capsule (reference electrode) in response to presentation of near-field acoustic stimuli. B, Example traces from control and mutant flies in response to synthetic pulse song stimulus. Individual responses to 10 consecutive stimuli are depicted as thin gray lines and their average as the thicker blue (control), orange (mutant), or magenta (mutant) line, in agreement with the color scheme in C. C, Scatterplot of SEP amplitudes recorded from flies with Shal-related genotypes. Smaller delayed responses represent acoustic echo artifacts. Each dot represents the SEP amplitude recorded from one antenna, and the number of antennae tested for each genotype is indicated at the bottom of the graph. Bars indicate means, and error bars represent SEM. Controls (blue dots) and the Shal protein trap flies, ShalMI00446-GFSTF.1 (green dots), are not significantly different, but all other genotypes are significantly different from controls. Strong alleles (orange dots) produce significantly lower SEPs than weak alleles (magenta dots). The dominant-negative Shal genotype (ShalDN = w elavC155-Gal4;; UAS-HA-ShalW362F/TM6B, Tb Hu) behaves as a strong mutant genotype. Brown–Forsythe ANOVA; p < 0.0001; with Dunnett's multiple comparisons (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). For the map of the Shal locus and insertional constructs of Shal alleles, see Extended Data Figure 2-1.
- Figure 3.
Shal loss of function shifts antennal resonant frequency with little effect on power gain. A, LDV preparation. Reflections from a laser beam focused on the arista allow precise recording of antennal movements. In the awake state, the antenna of a control fly (blue trace) shows vibrations in a range of frequencies below 1.5 kHz, with a peak at ∼240 Hz (vertical blue line). The same antenna under CO2 sedation (light blue trace) shows lower magnitude vibrations with a peak in the 800 Hz range (light blue dashed vertical line). These recordings in the absence of sound stimuli are called “FF.” Similar laser vibrometry recordings from an awake Shalf00495 mutant (orange trace) shows peak vibrations in the 400 Hz range (orange vertical line), but when sedated, vibrations from the same fly (light orange trace) are in the 800 Hz range (light orange dashed vertical line) resembling a sedated control fly. B, Scatterplots of the peaks (best frequencies) of antennal FF in the awake state (left graph) and the sedated state (right graph). Each dot represents the best frequency of one antenna, and the number of antennae tested for each genotype is indicated at the bottom of each graph. Bars indicate means; error bars represent SEM. Colors of dots match genotypes of Figure 2. In the awake state, the strong alleles (orange dots) show best frequencies significantly higher than controls (blue dots). However, the weak alleles (magenta dots) as well as the Shal protein trap (green dots) do not significantly shift the best frequencies compared with controls. Brown–Forsythe ANOVA; p < 0.0001; with Dunnett's multiple comparisons (ns, not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001). In the sedated state, none of the genotypes significantly differ from controls. C, A scatterplot of estimated power gain calculations. Genotypes and dot colors as in B. Power gains in Shal mutant genotypes do not differ significantly from controls. Kruskal–Wallis ANOVA with Dunn's multiple comparisons (ns, not significant). For plots of Q values of LDV data for Shal genotypes, see Extended Data Figure 3-1.
- Figure 4.
Model of Shal in active mechanotransduction in JO. A, During sensory transduction, sound-induced movements of the arista are transferred by the dendritic caps (blue) to the JO neuron ciliated dendrites (red) to initiate mechanotransduction. In the absence of sound stimuli, active ciliary movements in the sensory dendrites of JO neurons transfer kinetic energy to the antennal joint via the dendritic cap resulting in antennal vibrations (FF). B, In the JO neurons, several ion channels have been localized to the dendritic compartment. NompC (TRPN) is localized in the distal-most ciliary compartment beyond the ciliary dilation. This ciliary segment is nonmotile given the absence of axonemal dynein arms. Nan/Iav (TRPV) channels localize in the motile proximal ciliary segment, colinear with the localization of axonemal dynein arms. In this study, we show that Shal localizes at low intensity along the entire sensory cilium and strongly in the neuron soma membrane. C, Schematic of complex interplay between the dynamics of membrane currents (receptor potentials) mediated by both TRPN and TRPV and the timing of active ciliary movements. Ciliary localization of Shal is consistent with a role in development of receptor potentials and directly or indirectly affecting motor activity. Alternatively, rather than dynamic cycle-by-cycle gating, we cannot rule out the possibility that Shal provides a static bias in membrane potential that impacts TRPN- and TRPV-mediated currents and active ciliary movements. Either way, loss of Shal may change the receptor potential dynamics sufficiently to shift the motor activity timing, shifting the antennal tuning. Altered tuning together with altered receptor potentials may reduce the activation of action potentials at the axon. Localization of Shal in the inner dendritic segment and in the neuron soma may affect the propagation of the sensory receptor potential or its conversion into an action potential. Some alleles may affect this somatic function, disrupting the generation or propagation of full action potentials without affecting active ciliary movements.
Tables
Line Research resource identifier References Canton S (Hotta) w1118; PBac{WH}Shalf00495 RRID: BDSC_18338 (Thibault et al., 2004) w1118; Mi{ET1}ShalMB05249 RRID: BDSC_24326 (Metaxakis et al., 2005; Bellen et al., 2011) y1 w*; Mi{MIC}ShalMI00446/TM3, Sb RRID: BDSC_31006 (Venken et al., 2011) y1 w*; Mi{MIC}ShalMI10881 RRID: BDSC_56089 (Venken et al., 2011) y1 w*; Mi{PT-GFSTF.1}ShalMI00446-GFSTF.1 RRID: BDSC_60149 (Nagarkar-Jaiswal et al., 2015) w elavC155-Gal4;; UAS-HA-ShalW362F/TM6B, Tb Hu (Ping et al., 2011) Genotype N Mean SD Mean difference from control 95% confidence intervals of difference SEPs (Fig. 2C) Control 56 913.9 126.8 ShalMI00446-GFSTF.1 9 974.4 72.7 60.6 13.3–126 ShalMI00446 9 332.4 45.7 −581 −630 to −543 Shalf00495 8 241.1 31.8 −673 −715 to −638 ShalDN 10 299.6 61.3 −614 −668 to −570 ShalMI10881 8 425.8 36.9 −488 −534 to −453 ShalMB05249 10 390.2 38.0 −524 −566 to −487 Arista best freq. (awake; Fig. 3B) Control 25 240.9 45.5 ShalMI00446-GFSTF.1 10 273.3 61.5 32.4 2.2–88 ShalMI00446 17 340.9 36.1 100 74.0–123 Shalf00495 11 423.7 52.5 183 151–219 ShalDN 11 338.9 78.2 98.1 57.4–150 ShalMI10881 10 197.0 78.2 −43.9 −83.5 to 18.4 ShalMB05249 10 260.6 88.0 19.7 −21.5 to 98.2 Arista best freq. (sedated; Fig. 3B) Control 25 797.9 94.0 ShalMI00446-GFSTF.1 10 827.5 83.6 29.6 −22.2 to 106 ShalMI00446 17 873.1 68.1 75.2 25.9–122 Shalf00495 11 758.0 84.2 −39.9 −90.4 to 26.6 ShalDN 11 737.7 29.0 −60.1 −99.5 to −21.1 ShalMI10881 10 856.0 155 58.1 −36.9 to 161 ShalMB05249 10 796.3 80.9 −1.5 −60 to 56.6 Power gain (Fig. 3C) Control 25 7.32 7.9 ShalMI00446-GFSTF.1 10 13.6 20.3 6.3 −1.7 to 28 ShalMI00446 17 20.1 25.0 12.8 3.7–30 Shalf00495 11 2.61 2.8 −4.7 −8.6 to −1.6 ShalDN 11 2.82 2.1 −4.9 −8.5 to −1.6 ShalMI10881 10 6.43 4.8 −0.9 −5.2 to 3.1 ShalMB05249 10 2.60 4.1 −4.7 −8.4 to −0.7 Q (awake; Extended Data Fig. 3-1A) Control 25 1.20 0.43 ShalMI00446-GFSTF.1 10 1.80 0.46 0.60 0.31–0.96 ShalMI00446 17 2.66 1.74 1.46 0.83–2.54 Shalf00495 11 0.68 0.17 −0.52 −0.75 to −0.36 ShalDN 11 0.43 0.13 −0.77 −1.00 to −0.62 ShalMI10881 10 1.47 2.02 0.28 −0.45 to 2.39 ShalMB05249 10 1.26 0.64 0.06 −0.25 to 0.62 Q (sedated; Extended Data Fig. 3-1B) Control 25 0.96 0.20 ShalMI00446-GFSTF.1 10 0.94 0.23 −0.02 −0.15 to 0.15 ShalMI00446 17 1.02 0.26 0.06 −0.07 to 0.19 Shalf00495 11 1.00 0.17 0.04 −0.08 to 0.14 ShalDN 11 1.01 0.18 0.05 −0.11 to 0.15 ShalMI10881 10 1.04 0.21 0.08 −0.05 to 0.23 ShalMB05249 10 1.11 0.20 0.15 −0.01 to 0.26
Figure 1-1
Expression of Shal in antennal single nucleus RNA sequencing (Fly Cell Atlas) A. Annotated clustering of single-nucleus RNA transcript expression from antenna (reproduced from Li et al. (2022) with permission), showing a cluster of cells representing the JO neurons (circled). B. Expression of Shal (red) depicted over the same clusters indicates that Shal is expressed in JO neurons (circled) as well as olfactory neurons, using SCope (https://scope.aertslab.org/) (Davie et al., 2018). Download Figure 1-1, TIF file.
Figure 2-1
Map of Shal locus. Upper panel shows a screenshot of the JBrowse genome browser depicting the Shal locus on chromosome 3L. Shal is transcribed in the leftward direction, with three transcript splice isoforms (coding regions in orange boxes, non-coding regions in gray). Transposon insertion sites are depicted by small blue triangles, labeled. Corresponding transposon structures are diagrammed below (from the Gene Disruption Project (https://flypush.research.bcm.edu/pscreen/transposons.html)), with orientation information relative to the map. Download Figure 2-1, TIF file.
Figure 3-1
Q factors of LDV data for Shal genotypes. Scatter plots of the Q values, indicating sharpness of the peaks, of antennal free fluctuations from Fig. 3 in the awake state (A) and the sedated state (B). Each dot represents the Q of one antenna recording and the number of antennae tested for each genotype is indicated at the bottom of each graph. Bars indicate means; error bars represent SEM. Colors of dots match genotypes of Fig. 2 and 3. In the awake state, the strong alleles (orange dots) show statistically significantly differences from controls (blue dots). However, the weak alleles (magenta dots) as well as the Shal protein trap (green dots) do not significantly shift the Q values compared to controls. Kruskal-Wallis ANOVA, p < 0.0001, p = 0.32 for sedated flies, with Dunn’s multiple comparisons (ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001). In the sedated state, none of the genotypes significantly differs from controls (Kruskal-Wallis, p = 0.32). Download Figure 3-1, TIF file.
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