Co-expression of <i>Gr64b</i> and <i>Gr64e</i> confers glycerol responsiveness.

<p>(A and B) Representative traces (A) and response frequencies (B) from S6 sensilla in <i>norpA</i>^<i>P24</i> flies elicited by 1% HxA and 10% glycerol solutions. n = 5–10. **p < 0.001 (Mann-Whitney <i>U</i> test). (C) Labellar PER responses to glycerol in <i>norpA</i>^<i>P24</i> flies. A 5% glycerol solution was used. n = 5–7. (D and E) Representative traces (D) and response frequencies (E) from the indicated sensilla of <i>Gr64af</i> flies co-expressing <i>Gr64b</i> and <i>Gr64e</i> in sweet GRNs elicited by 10% glycerol. n = 5–13. **p < 0.001 (Kruskal-Wallis with Mann-Whitney <i>U post-hoc</i> tests). (F and G) Representative traces (F) and response frequencies (G) from L6 sensilla of <i>Gr64af</i> flies co-expressing <i>Gr64b</i> and <i>Gr64e</i> in low salt-sensing GRNs elicited by 10% glycerol. n = 4–20. *p < 0.01, **p < 0.001 (Kruskal-Wallis with Mann-Whitney <i>U post-hoc</i> tests). (H) Labellar PER responses to glycerol in <i>Gr64af</i> flies co-expressing <i>Gr64b</i> and <i>Gr64e</i> using <i>Gr5a</i>-<i>GAL4</i>. A 5% glycerol solution was used. n = 3–9. **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). Data are presented as medians and quartiles (B, E, and G) or as means ± SEM (C and H).</p

<i>Gr64e</i> is required for electrophysiological responses to HxA.

<p>(A) Electrophysiological response profiles of labellar sensilla to 1% HxA. Representative traces are shown above and action potential frequencies in the indicated sensilla are shown below. n = 3–25. (B and C) Representative traces from S6 sensilla (B) and response frequencies (C) evoked by 1% HxA in the indicated genotypes. n = 5–11. *p < 0.01 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). (D and E) Testing whether <i>Gr64e</i> is required for HxA-evoked responses. Representative traces (D) and response frequencies (E) from S6 sensilla evoked by 1% HxA. We expressed a <i>Gr64e</i> cDNA in <i>Gr64e</i>^<i>LEXA</i> flies or <i>Gr64af</i> flies using <i>Gr5a</i>-<i>GAL4</i>. n = 7–10. **p < 0.001 (Kruskal-Wallis with Mann-Whitney <i>U post-hoc</i> tests). Data are presented as medians with quartiles (A, C, and E).</p

Functional redundancy between GR64e and TRPA1 downstream of PLC.

<p>(A and B) Representative traces (A) and response frequencies (B) from S6 and L3 sensilla of <i>Gr64af</i> flies expressing <i>TrpA1(A)10a</i> in sweet GRNs, as evoked by 1% HxA and 10% glycerol. n = 5–10. **p < 0.001 (Kruskal-Wallis with Mann-Whitney <i>U post-hoc</i> tests). (C) PER analysis to determine whether expression of <i>TrpA1(A)10a</i> under the control of <i>Gr5a</i>-<i>GAL4</i> rescues the <i>Gr64af</i> defect in FA sensing. Solutions of 0.4% HxA and 5% glycerol were used. n = 5–11. **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). (D and E) Representative traces (D) and response frequencies (E) of S2 sensilla responding to 1 mM NMM and S6 sensilla responding to 1 mM ARI, all from <i>TrpA1</i>^<i>1</i> mutant flies expressing <i>Gr64e</i>. n = 8–21. **p < 0.001 (Kruskal-Wallis with Mann-Whitney <i>U post-hoc</i> tests). Data are presented as medians and quartiles (B and E) or as means ± SEM (C).</p

<i>Gr64e</i> is required for fatty acid sensing.

<p>(A) PER screening for individual <i>Gr64</i> cluster genes required for HxA sensing. A 0.4% HxA solution was used. <i>norpA</i>^<i>P24</i> was included as a positive control. n = 6–11. *p < 0.01, **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). (B) Testing whether <i>Gr64c</i> is required for labellar PER responses to HxA. To test the rescue of the <i>Gr64c</i>^<i>LEXA</i> phenotype, we expressed a <i>Gr64c</i> cDNA in the <i>Gr64c</i>^<i>LEXA</i> background using <i>Gr5a</i>-<i>GAL4</i>. 0.4% HxA, 5% Gly, and 100 mM Suc solutions were used. n = 6–9. **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). (C) PER analysis to determine whether <i>Gr64e</i> is required for labellar PER responses to glycerol and HxA. We expressed a <i>Gr64e</i> cDNA in <i>Gr64e</i>^<i>LEXA</i> flies using <i>Gr5a</i>-<i>GAL4</i>. 0.4% HxA and 5% Gly solutions were used. n = 6–10. *p < 0.01, **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). (D) PER analysis to determine whether <i>Gr64e</i> is required for labellar PER responses to various FAs. 0.4% FAs were used. n = 4–8. **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). (E) Rescue of the <i>Gr64af</i> defect in HxA sensing by expressing <i>Gr64e</i> under the control of <i>Gr5a</i>-<i>GAL4</i>. A 0.4% HxA solution was used. n = 6–14. **p < 0.001 (one-way ANOVA with <i>post</i>-<i>hoc</i> Tukey tests). All data are presented as means ± SEM.</p

Models for activation of GR64e in fatty acid sensing and glycerol sensing.

<p>(A) Schematic model for GR64b and GR64e functioning as a ligand-gated channel in glycerol sensing. (B) Model for activation of GR64e in FA sensing. Activation of an unknown FA receptor stimulates phospholipase C (PLC), thereby activating GR64e.</p

The <i>Gr64</i> cluster is required for fatty acid sensing.

<p>(A) Schematics of the <i>Gr64</i> cluster locus and the strategy for generating <i>Gr64af</i> using the CRISPR/Cas9 system. The scissors indicate the guide RNA targeting sites cut by Cas9 and the bent arrows indicate the regions where excision occurred. The arrow heads indicate the primers used for deletion validation. Genomic PCR is shown on the right. (B) Labellar PER responses to various sugars in <i>Gr64af</i> flies. 100 mM sucrose (Suc), 500 mM glucose (Glu), 100 mM fructose (Fru), 500 mM trehalose (Tre), and 5% glycerol (Gly) solutions were used. n = 9. **p < 0.001 (unpaired Student’s <i>t</i>-test). (C) Labellar PER response to low salt (50 mM NaCl) in <i>Gr64af</i> flies. n = 8. *p < 0.01 (unpaired Student’s <i>t</i>-test). (D) Optogenetic activation of sweet GRNs in two groups, <i>Gr5a</i>><i>ReaChR</i> (control) and <i>Gr5a</i>><i>ReaChR</i>;<i>Gr64af</i> (<i>Gr64af</i>), with retinal (+) and without retinal (-). n = 7–10. (E) Labellar PER responses to various FAs in <i>Gr64af</i> flies. 0.4% solutions of hexanoic acid (HxA), octanoic acid (OcA), oleic acid (OA), and linoleic acid (LA) were used. n = 8. **p < 0.001 (unpaired Student’s <i>t</i>-test). (F) Labellar PER responses to HxA in the indicated genotypes. A 0.4% HxA solution was used. <i>n</i> = 5–11. All data are presented as means ± SEM.</p

Immunohistochemical (IHC) staining of permanent and deciduous PDL tissues (A–P).

<p>(<b>A</b>, <b>E</b>) IHC staining for IGF2BP1 in permanent and (<b>I</b>, <b>M</b>) deciduous PDL tissues. (<b>B</b>, <b>F</b>) IHC staining for IL6 in permanent and (<b>J</b>, <b>N</b>) deciduous PDL tissues. (<b>C</b>, <b>G</b>) IHC staining for LRRTM1 in permanent and (<b>K</b>, <b>O</b>) deciduous PDL tissues. (<b>D</b>, <b>H</b>) IHC staining for ADAMTSL3 in permanent PDL tissues and (<b>L</b>, <b>P</b>) deciduous PDL tissues. Abbreviations: pe-PDL, permanent PDL tissues; de-PDL, deciduous PDL tissues. Scale bars: 100 µm in <b>A–D</b> and <b>M–P</b>; 20 µm in <b>E–H</b> and <b>I–L</b>.</p

Main categories of genes expressed specifically in deciduous PDL tissues (de-PDL) and permanent PDL tissues (pe-PDL) on the basis of their biological processes.

<p>Numbers of involved genes are listed in <i>x</i>-axis, F-statistic <i>p</i><0.05.</p

Up-regulated genes in permanent PDL tissue (compared to deciduous PDL tissue).

<p>Up-regulated genes in permanent PDL tissue (compared to deciduous PDL tissue).</p

Main categories of genes expressed specifically in de-PDL and pe-PDL on the basis of their molecular functions.

<p>Numbers of involved genes are listed in the pie charts, F-statistic <i>p</i><0.05.</p