Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila
- Brandeis University, United States
- University of Lausanne, Switzerland
- Harvard University, United States
- University of Miami, United States
Figures
Figure 1
IR93a is expressed in Dorsal Organ Cool Cells (DOCCs) and is required for cool avoidance.
(a) Gene structure of the Ir93a locus; sequences encoding the transmembrane (TM) domains and channel pore are colored. The blue triangle denotes site of MiMIC insertion in Ir93aMI05555, and the CRISPR/Cas9-generated deletion in the Ir93a122 allele is shown below. (b) Schematic of the larval anterior showing the bilaterally symmetric Dorsal Organ Ganglia (grey) within which three Dorsal Organ Cool Cells (DOCCs) are located. (c) Immunofluorescence of the larval anterior (corresponding to the boxed region in the schematic) showing expression of IR93a protein (magenta) in DOCCs (Ir21a-Gal4;UAS-GFP [Ir21a>GFP]) (green), as well as additional sensory neurons. Ir93aMI05555 mutants lack IR93a immunostaining. The arrow and arrowhead label the soma and dendritic bulb of one of the DOCCs. Scale bar is 10 µm. (d) Cool avoidance behavior assessed as navigational bias (movement toward warmth / total path length) of individual larval trajectories on an ~0.36°C/cm gradient extending from ~13.5°C to ~21.5°C, with a midpoint of ~17.5°C. Letters denote statistically distinct categories (alpha = 0.05; Tukey HSD). wild type (Canton-S), n = 37 animals. Ir93aMI05555, n = 132. Ir21a-Gal4/+; Ir93aMI05555, n = 72. Ir93aMI05555,UAS-Ir93a/ Ir93aMI05555, n = 80. Ir21a-Gal4/+; Ir93aMI05555,UAS-Ir93a/ Ir93aMI05555, n = 45. Ir93a122, n = 101.
Figure 2
Cool-responsive calcium and voltage changes in DOCCs require IR93a.
(a) Left: DOCC responses monitored using R11F02>GCaMP6m. DOCC cool-responsive increases in fluorescence are dramatically reduced in Ir93aMI05555, and responses are rescued by expression of a wild-type Ir93a cDNA under R11F02-Gal4 control. Traces, average ± SEM. Right: Ratio of fluorescence at 14°C versus 20°C depicted using a violin plot (internal white circles show median; black boxes denote 25th to 75th percentiles; whiskers extend 1.5 times interquartile range). Letters denote statistically distinct categories, p<0.01, Steel-Dwass test. wild type, n = 12 cells. Ir93aMI05555, n = 44. Ir93aMI05555; R11F02>Ir93a, n = 46. (b) Temperature-dependent DOCC voltage responses in the sensory endings of wild-type (upper panels) or Ir93aMI05555 mutant (lower panels) larvae monitored using R11F02>Arclight. Arrowheads denote DOCC dendritic bulbs. Note that Arclight fluorescence decreases upon depolarization. Asterisks denote cuticular autofluorescence from adjacent sensory structures. (c) Robust cool-responsive depolarization of DOCC sensory endings is observed in otherwise wild-type animals using either R11F02>Arclight or Ir21a>Arclight. Depolarization response is eliminated in Ir93aMI05555, Ir25a2, and Ir21a∆1 mutants. Traces, average ± SEM. Violin plot depicts ratio of fluorescence at 14°C versus 20°C. ** denotes distinct from wild-type control, p<0.01 compared to control, Steel-Dwass test. R11F02-Gal4;UAS-Arclight, n = 57 cells. R11F02-Gal4;UAS-Arclight;Ir93aMI05555, n = 24. R11F02-Gal4;UAS-Arclight; Ir25a2, n = 30. Ir21a-Gal4;UAS-Arclight, n = 18. Ir21a-Gal4;UAS-Arclight; Ir21a∆1, n = 23.
Figure 3
IR93a is co-expressed with IR25a and IR40a in sacculus neurons.
(a) Left: schematic of the adult Drosophila antenna, illustrating the location of the sacculus (red) in the interior of this appendage. Right: the sacculus is composed of three main chambers (I, II, III), which are lined with sensilla of various morphologies (cartoon adapted from [Shanbhag et al., 1995]). (b) Top: immunofluorescence on a whole-mount wild-type antenna showing expression of IR93a protein (green) in two groups of soma (arrows) around sacculus chambers I and II; these chambers are visualized by cuticle autofluorescence shown in the images on the right. The arrowhead marks the concentration of IR93a in the dendritic endings that innervate the sensilla in chamber I. Note that the dendrites of chamber II neurons are not visible in this image; sensilla localization of IR93a in these cells is more easily detected in antennal sections; see panel (d). Bottom: Ir93aMI05555 mutants lack detectable IR93a protein. Scale bar is 20 µm. (c–e) Double immunofluorescence with the indicated antibodies on antennal cryosections revealing co-expression of these IRs in sacculus neurons; the arrows point to the cluster of neurons innervating chamber II. Scale bar is 10 µm. IR25a is expressed in additional neurons that do not express IR93a or IR40a because of IR25a’s broader role as an olfactory IR co-receptor (Abuin et al., 2011).
Figure 4 with 1 supplement
Hygrosensory behavior requires IR93a, IR25a and IR40a.
(a) Schematic of the hygrosensory behavior assays. ~67% to ~96% RH gradients were generated by filling wells with either a saturated solution of ammonium nitrate in water or pure water. ~89% to ~96% RH gradients were generated by pairing empty wells with wells filled with pure water. Nylon mesh prevented fly contact with solutions. Dry preference was quantified by counting flies on either side of chamber midline. 25–35 flies were used per assay. (b) Mean ± SD of RH and temperature measured at indicated gradient positions. ~67% to ~96% RH, n = 58 gradients. ~89% to ~96% RH, n = 28. (c,d) Dry preference assessed on ~67% vs. ~96% (c) and ~89% vs. ~96% (d) gradients. Asterisks denote statistically distinct from wild type (**p<0.01; *p<0.05, Steel with control). wild type, n = 16 assays. Ir8a mutant (Ir8a1), n = 8. Ir76b mutant (Ir76b2), n = 14. Ir21a mutant (Ir21a123), n = 14. Ir25a mutant (Ir26a2), n = 11. Ir25a rescue (Ir25a2; UAS-Ir25a), n = 15. Ir40a mutant (Ir40a1), n = 15. Ir40a rescue (Ir40a1; UAS-Ir40a), n = 9. Ir40a CRISPR mutant (Ir40a134), n = 10. Ir93a mutant (Ir93aMI05555), n = 11. Ir93a rescue (Ir93aMI05555, UAS-Ir93a), n = 14. Ir40a mutant alleles and thermosensory behavior are shown in Figure 4—figure supplement 1a–b. Note that UAS-cDNA rescues were observed in the absence of Gal4 drivers, reflecting Gal4-independent expression of UAS transgenes (Figure 4—figure supplement 1c–d).
Figure 4—figure supplement 1
Description of Ir40a mutants and analysis of Gal4-independent transgene expression.
(a) Gene structure and sequence alterations in Ir40a alleles. Regions encoding transmembrane domains (TMs) and pore region are in red. The Ir40a promoter region present in Ir40a-Gal4 is indicated in green. (b) Larval cool avoidance behavior (assayed as in Figure 1d) is unaffected by mutation of Ir40a. wild type, n = 37 animals. Ir40a1, n = 55. (c) RT-PCR analysis of Gal4-independent expression of UAS-Ir transgenes in adult heads in the indicated genotypes. Upper panels, IR-specific RT-PCR products. Lower panels: RpL32 (a ribosomal protein gene) as a cDNA synthesis control. Asterisk indicates a background amplification product observed in some Ir25a PCR reactions. The mechanism underlying Gal4-independent UAS-transgene expression is unknown, but is a phenomenon that has been previously reported (Mao et al., 2014). (d) Top: IR25a protein expression in the sacculus of wild-type, Ir25a2 and Ir25a2;UAS-Ir25a animals. Bottom: IR93a protein expression in the sacculus of wild-type, Ir93aMI05555 and Ir93aMI05555;UAS-mCherry:IR93a animals. Gal4-independent expression of UAS transgenes restores expression of IR25a and IR93a in the dendrites of sacculus neurons (arrowhead).
Figure 5
IR-dependent physiological responses to dry air.
(a) Schematic of the Drosophila head (viewed from above) illustrating the projection of IR40a/IR93a/IR25a-expressing neurons (green) (labeled using Ir40a-Gal4 [Silbering et al., 2011]) from the sacculus to the antennal lobes in the brain, visualized through a hole in the head cuticle. (b) Raw fluorescence image of Ir40a axons (in Ir40a-Gal4;UAS-GCaMP6m animals) innervating the arm and column in the antennal lobe. The dashed circle indicates the position of the ROI used for quantification in panels (d–g). (c) Color-coded images (reflecting GCaMP6m fluorescence intensity changes) of IR40a neuron responses to a switch from 90% to 7% RH ('Dry response') and to a switch from 7% to 90% RH ('Moist response'). (d,f) Moisture-responsive fluorescence changes in the arm (moist = 90% RH, dry = 7% RH). Traces represent average ± SEM. (e,g) Quantification of changes in ∆F/F (mean fluorescence change in the ROI shown in [b]) upon shift from moist to dry (e) or dry to moist (g). Dry responses were quantified as [∆F/F at 7% RH (average from 4.5 to 6.5 s after shift to 7% RH)] - [∆F/F at 90% RH (average from 3.5 to 1 s prior to shift to 7% RH)], and moist responses quantified by performing the converse calculation. Genotypes: control: n = 17 animals (pooled data from Ir40a-Gal4,Ir40a1/IR40a-Gal4,+;UAS-GCaMP6m/+, n = 9; IR40a-Gal4;UAS-GCaMP6m,Ir93aMI05555/+, n = 8). Ir93a mutant (Ir40a-Gal4;UAS-GCaMP6m,Ir93aMI05555/Ir93aMI05555), n = 10. Ir93a rescue (Ir40a-Gal4;UAS-GCaMP6m,Ir93aMI05555/UAS-mcherry:Ir93a,Ir93aMI05555), n = 8. Ir40a mutant (Ir40a-Gal4,Ir40a1;UAS-GCaMP6m/+), n = 8. Ir40a rescue (Ir40a-Gal4,Ir40a1;UAS-GCaMP6m/UAS-Ir40a), n = 6. **p<0.01, distinct from controls and rescues, Steel-Dwass test.
Figure 6
The TRP channels Nanchung and Water witch do not mediate IR-dependent dry sensation.
(a) Dry preference assessed on ~67% to ~96% gradient. Asterisks denote statistically different responses from wild type (**p<0.01; Steel with control). wild type, n = 16 assays. nan mutant (nan36a), n = 9. wtrw mutant (wtrw2), n = 9. (b–e) Moisture-responsive fluorescence changes of IR40a neurons recorded and quantified as described in Figure 5. Genotypes: control: n = 5 animals (Ir40a-Gal4,UAS-GCaMP6m/+). nan mutant (Ir40a-Gal4,UAS-GCaMP6m/+;nan36a), n = 5. wtrw mutant (Ir40a-Gal4,UAS-GCaMP6m/+ ;wtrw2), n = 7. (All P>0.4 versus control, Steel with control).
Author response image 1
Ammonia increases the evaporative cooling effect of water.A temperature probe (Almemo Pt100, ZA9030-FS2) was dipped into water or 3% ammonia and immediately removed.
The temperature of the probe was recorded at 1 Hz during 10 s before and 50 s after dipping. The arrowhead indicated the time of dipping into the solution. Traces show temperature changes (mean±SEM; n = 10-11) relative to the mean room temperature before dipping. The drop in temperature reflects evaporative cooling. This phenomenon is more pronounced after dipping the probe in ammonia than in water.
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Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila
eLife 5:e17879.
https://doi.org/10.7554/eLife.17879
