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Ionotropic Receptor-dependent moist and dry cells control hygrosensation in Drosophila

  1. Zachary A Knecht
  2. Ana F Silbering
  3. Joyner Cruz
  4. Ludi Yang
  5. Vincent Croset
  6. Richard Benton  Is a corresponding author
  7. Paul A Garrity  Is a corresponding author
  1. Brandeis University, United States
  2. University of Lausanne, Switzerland
Research Advance
Cite this article as: eLife 2017;6:e26654 doi: 10.7554/eLife.26654
4 figures and 1 additional file

Figures

Figure 1 with 1 supplement
Ir68a and Ir40a reporters are expressed by neighboring neurons in the Drosophila sacculus.

(a) Left: schematic of the adult Drosophila antenna, showing the location of the sacculus (red) inside the antenna. Right: the sacculus contains three chambers (I, II, III) lined with sensilla of various morphologies (modified from Shanbhag et al. [1995]) and Knecht et al. [2016]). (b) Immunostaining of the antenna of a Ir68a-Gal4/UAS-myr:GFP (Ir68a>GFP) fly (left). Ir68a is expressed in neurons that send processes to sacculus chamber II (9.8 ± 0.4 neurons (mean ± SEM); n = 8 antennae). Arrowheads denote sensory endings. Scale bar in all panels indicates 10 µm. Cuticle autofluorescence outlines sacculus chambers in the right panel. (c) Immunostaining of LexAop-RFP(II);Ir68a-Gal4/Ir40a-LexA,UAS-myr:GFP (Ir68a>GFP, Ir40a>RFP) flies reveals non-overlapping expression in cells adjacent to chamber II. Arrows indicate select cell bodies. Arrowhead marks an Ir68a>GFP-labeled dendrite projecting into a chamber II sensillum. Cuticle autofluorescence outlines sensilla of the sacculus in the GFP channel. (d,e) Immunofluorescence on antennal cryosections reveals overlapping expression of Ir68a>GFP with IR25a protein (d) and IR93a protein (e) in sacculus neurons. Yellow arrows denote cells that detectably co-express Ir68a>GFP and IR25a (d) or Ir68a>GFP and IR93a (e). Purple arrows denote cells expressing only IR25a or IR93a, reflecting their broader expression in the sacculus, including co-expression with IR40a (Knecht et al., 2016). (fg) Ir68a>GFP-labeled and Ir40a>RFP-labeled axons project to distinct regions of the antennal lobe. In panel g, the antennal lobe neuropil is labeled using nc82. (h) Schematic indicating the relative position of Ir40a neuron and Ir68a neuron projections; VP1 and VP4 glomerular nomenclature is from Grabe et al. (2015). The Ir68a>GFP-labeled glomerulus (which has not been previously noted) has been denoted VP5 to maintain consistency with existing nomenclature. D-dorsal; V-ventral; L-lateral; M-medial. The organization of the Ir68a locus including the region used to generate Ir68a-Gal4 is provided in Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.26654.002
Figure 1—figure supplement 1
Organization of Ir68a locus.

Exons are illustrated as boxes; shaded regions correspond to the UTRs. Sequences encoding the transmembrane (TM) domains and channel pore of IR68a are colored. The dark blue triangles denote transposon insertion sites in Ir68ac04139 and Ir68aMB05565. The promoter region in Ir68a-Gal4 transgene is indicated in green. Sequences included in the Ir68a+ rescue transgene are indicated by the light blue bar.

https://doi.org/10.7554/eLife.26654.003
Ir68a is required for humidity detection by moist cells.

(a) Schematic of the Drosophila head (viewed from above) illustrating the projection of Ir68a-Gal4-labeled neurons (green) from the sacculus to the antennal lobes in the brain, visualized through a hole cut in the head cuticle. (b) Left panel: Raw fluorescence image of Ir68a-labeled axons (in an Ir68a-Gal4;UAS-GCaMP6m(III) animal) innervating the antennal lobe. The circle indicates the position of the ROI used for quantification. Middle and right panels: color-coded images (reflecting GCaMP6m relative fluorescence intensity changes) of responses to a switch from 7% to 90% RH (‘Moist response’) and to a switch from 90% to 7% RH (‘Dry response’). Scale bar is 10 µm. (c,d) Moist-elicited (c) and dry-elicited (d) fluorescence changes in the region of interest in panel (b) (moist = 90% RH, dry = 7% RH). Left panels: Traces represent mean ± SEM. Right panels: Quantification of responses. Letters denote statistically distinct groups (p≤0.05) Steel-Dwass. Data obtained using UAS-GCaMP6m(II) and UAS-GCaMP6m(III) were analyzed separately. Moist-responses were calculated as [F/F0 at 90% RH (average F/F0 from 4.5 to 6.5 s after shift to 90% RH)] - [F/F0 at 7% RH (average F/F0 from 3.5 to 1 s prior to shift to 90% RH)]. Dry-responses were quantified using the converse calculation. Violin plots: internal white circles show median; black boxes denote 25th to 75th percentiles; whiskers extend 1.5x the interquartile range. Genotypes: wild type (UAS-GCaMP6m(II)): Ir68a-Gal4,UAS-GCaMP6m(II) (n = 7 animals). Ir68a-/-: UAS-GCaMP6m(II);Ir68a-Gal4,Ir68ac04139/Ir68ac04139 (n = 9). Ir68a rescue: Ir68a+ rescue transgene(II)/UAS-GCaMP6m(II);Ir68a-Gal4,Ir68ac04139/Ir68ac04139 (n = 8). Ir93a-/-: UAS-GCaMP6m(II);Ir68a-Gal4,Ir93aMI05555/Ir93aMI05555 (n = 8). Ir93a rescue: UAS-GCaMP6m(II);Ir68a-Gal4,Ir93aMI05555/UAS-mCherry:Ir93a,Ir93aMI05555) (n = 6). wild type (UAS-GCaMP6m(III)): Ir68a-Gal4,UAS-GCaMP6m(III) (n = 10 animals). Ir25a-/-: Ir25a2;Ir68a-Gal4,UAS-GCaMP6m(III) (n = 8). Ir25a rescue: Ir25a2,Ir25aBAC/Ir25a2;Ir68a-Gal4,UAS-GCaMP6m(III) (n = 8). Ir40-/-: Ir40a1;Ir68a-Gal4,UAS-GCaMP6m(III) (n = 8). (e,f) Moist-elicited (e) and dry-elicited (f) fluorescence changes in Ir40a-Gal4-labeled dry receptors, as in panels c-d. Genotypes: wild type: Ir40a-Gal4/UAS-GCaMP6m (n = 9). Ir68a-/-: Ir40aGal4/UAS-GCaMP6m;Ir68ac04139 (n = 9). Ir68a mutant alleles and genomic rescue fragment are shown in Figure 1—figure supplement 1. Source data for summary graphs are provided in Figure 2—source data 1.

https://doi.org/10.7554/eLife.26654.004
Figure 2—source data 1

Calcium imaging results.

Calcium imaging results used to create Figure 2 summary graphs.

https://doi.org/10.7554/eLife.26654.005
Ir68a is required for hygrosensory behavior.

(a) Schematic of hygrotaxis assay.~67% to ~96% RH gradients were generated as described (Knecht et al., 2016). Dry preference was quantified by counting flies on either side of chamber midline. 25–35 flies were used per assay. (b) Dry preference of hydrated flies. Asterisks denote values that are statistically distinct from wild type (**p<0.01, Steel with control). wild type (n = 12 assays). Ir25a2 (n = 12). Ir93aMI05555 (n = 11). Ir40a1 (n = 15). Ir68ac04139 (n = 16). Ir68aMB05565 (n = 14). Ir68aMB05565 + rescue (Ir68a+ rescue transgene(II);Ir68aMB05565) (n = 12). Ir40a1;Ir68aMB05565 (n = 15). Source data for summary graph are provided in Figure 3—source data 1.

https://doi.org/10.7554/eLife.26654.006
Figure 3—source data 1

Hygrotaxis behavior data.

Primary hygrotaxis behavior data for Figure 3.

https://doi.org/10.7554/eLife.26654.007
Ir40a and Ir68a each contribute to hygrotaxis in dehydrated flies.

(a) Schematic of hygrotaxis assay using dehydrated flies. (b) Hygrotaxis behavior in dehydrated flies. Letters denote statistically distinct groups (p<0.01, Tukey HSD). Genotypes: wild type (n = 15 assays). Ir25a2 (n = 13). Ir93aMI05555 (n = 14). Ir40a1 (n = 12). Ir68aMB05565 (n = 11). Ir40a1;Ir68aMB05565 (n = 12). Source data for summary graph are provided in Figure 4—source data 1.

https://doi.org/10.7554/eLife.26654.008
Figure 4—source data 1

Hygrotaxis behavior data.

Primary hygrotaxis behavior data for Figure 4.

https://doi.org/10.7554/eLife.26654.009

Additional files

Source code 1

Code for Calcium Imaging analysis

https://doi.org/10.7554/eLife.26654.010

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