Skip to main content
. 2021 Mar 5;10:e63036. doi: 10.7554/eLife.63036

Figure 6. Pdm3 is required to distinguish Ir75a and Ir75b neuron fate.

(A) Immunofluorescence for IR75a and IR75b on whole-mount antennae of control (peb-Gal4,UAS-Dcr-2/+) and pdm3RNAi#2 (peb-Gal4,UAS-Dcr-2/+;UAS-pdm3HMJ21205/+) animals. Scale bar = 10 µm. The schematics on the right summarize the distribution of labeled neurons. (B) Immunofluorescence for GFP and IR75a on whole-mount antennae of control (peb-Gal4,UAS-Dcr-2/+;Ir75a-GFP/+) and pdm3RNAi#2 (peb-Gal4,UAS-Dcr-2/+;Ir75a-GFP/UAS-pdm3HMJ21205) animals. Scale bar = 10 µm. (C) Immunofluorescence for GFP and IR75b on whole-mount antennae of control (peb-Gal4,UAS-Dcr-2/+;Ir75b-GFP/+) and pdm3RNAi#2 (peb-Gal4,UAS-Dcr-2/+;Ir75b-GFP/UAS-pdm3HMJ21205) animals. Scale bar = 10 µm. (D) Quantification of the number of neurons that express Ir75a-GFP or Ir75b-GFP in the genotypes shown in (B–C). Comparisons to the controls are shown (pairwise Wilcoxon rank-sum two-tailed test and p-values adjusted for multiple comparisons with the Bonferroni method, **p<0.001). The increase in number of Ir75b-GFP labeled neurons in pdm3RNAi is lower than the increase in IR75b-expressing neurons, potentially because the transgenic reporter is not fully faithful in this genetic background. (E) Immunofluorescence for nc82 and GFP on whole-mount antennal lobes of control and pdm3RNAi#2 animals. Genotypes are as in (B–C). Scale bar = 20 µm. Quantification of phenotypes are shown on the right. (F) Quantification of the number of Ir75a and Ir75b neurons in animals (peb-Gal4,UAS-Dcr-2/+;pdm3RNAi#2/+;UAS-Gal80ts/+) in which peb-Gal4-driven pdm3RNAi is continuously suppressed (19°C, the permissive temperature for the Gal4 inhibitor Gal80ts), continuously allowed (29°C, the restrictive temperature for Gal80ts) or induced only in adults (19°C → 29°C temperature shift after eclosion). Comparisons between conditions are shown for each neuron type (pairwise Wilcoxon rank-sum two-tailed test with Bonferroni correction for multiple comparisons, **p<0.001, NS p>0.05). (G) Immunofluorescence for IR75a and IR75b on whole-mount antennae of the genotypes shown in (F). Single optical sections are shown, to reveal the weak co-expression of IR75a and IR75b in a subset of cells (arrowheads). Scale bar = 10 µm. (H) Quantification of the number of neurons that co-express IR75a and IR75b. Comparisons to the controls are shown (pairwise Wilcoxon rank-sum two-tailed test with Bonferroni correction for multiple comparisons, **p<0.001, NS p>0.05). (I) Immunofluorescence for nc82 and GFP on whole-mount antennal lobes of pdm3RNAi#2 animals (peb-Gal4,UAS-Dcr-2/+;UAS-pdm3HMJ21205/Ir75b-GFP;UAS-Gal80ts/+) in which RNAi is allowed throughout development (29°C) or limited only to adults (19°C → 29°C temperature shift after eclosion). Quantification of glomerular labeling pattern by the Ir75b-GFP reporter is shown on the right of each image.

Figure 6.

Figure 6—figure supplement 1. Phenotypic analysis of Ir75a and Ir75b neurons with late developmental induction of pdm3 RNAi.

Figure 6—figure supplement 1.

(A) Quantification of the number of Ir75a and Ir75b neurons in control (Ir8a-Gal4/+) and pdm3RNAi#2 (Ir8a-Gal4/UAS-pdm3HMJ21205) animals. Comparisons to the control are shown for each neuron type (pairwise Wilcoxon rank-sum two-tailed test, **p<0.001, NS p>0.05). Although the number of IR75a-expressing cells was unchanged compared to controls, receptor protein levels were lower in several cells (see (B)). (B) Immunofluorescence for IR75a and IR75b on whole-mount antennae of control and pdm3RNAi#2 animals (genotypes as in (A)). Single optical sections are shown, to reveal the weak co-expression of IR75a and IR75b in a subset of cells (arrowheads). Scale bar = 10 µm. (C) Quantification of the number of neurons that co-express IR75a and IR75b. Comparison to the control is shown (pairwise Wilcoxon rank-sum two-tailed test). (D) Immunofluorescence for nc82 and GFP on whole-mount antennal lobes of pdm3RNAi#2 animals. Genotypes: Ir75a-GFP control (Ir75a-GFP/+;Ir8a-Gal4/+), Ir75a-GFP pdm3RNAi#2 (Ir75a-GFP/UAS-pdm3HMJ21205;Ir8a-Gal4/+), Ir75b-GFP control (Ir75b-GFP/+;Ir8a-Gal4/+), Ir75b-GFP pdm3RNAi#2 (Ir75b-GFP/UAS-pdm3HMJ21205;Ir8a-Gal4/+). Scale bar = 20 µm. Quantification of projection pattern phenotypes are shown on the right. In contrast to observations with adult-only pdm3 RNAi driven by peb-Gal4 (Figure 6I), we did not detect labeling of DP1l in these experiments. This difference is likely to reflect the weaker RNAi induced by Ir8a-Gal4 (and/or the absence of UAS-Dcr-2 in these genotypes), as is apparent from the quantification of Ir75a and Ir75b neuron numbers (compare panel (A) with Figure 6F).