Chromatin Modulatory Proteins and Olfactory Receptor Signaling in the Refinement and Maintenance of Fruitless Expression in Olfactory Receptor Neurons

Abstract

During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru). The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a) involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh). The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors.

Fig 1. fru-positive OR expression in at4 sensilla expands to developmentally related fru-negative ORNs in alh mutants.

A) Adult antennae and brains labeled with Or47bGal4 UAS-CD8GFP (green) in wild type and alh mutant clones. Magenta staining in brains is against N-cadherin, a neuropil marker. B) Adult antennae and brains labeled with Or88aGal4 UAS-CD8GFP in wild-type and alh mutant clones. C) Adult antennae and brains labeled with Or65aGal4 UAS-CD8GFP in wild-type and alh mutant clones. D–G) Quantification of cell bodies observed in the adult antennae of WT and alh mutant clones. For all graphs, asterisks indicate significant (p < .05) differences from wild type. Error bars represent standard error of the mean (SEM). An ANOVA was performed for each cell type and followed with Tukey’s Honest Significant Difference (HSD)—see Materials and Methods. D) Total Or47b-positive cells. Wild type flies were significantly different from both alh conditions (p < .0001). n = 10–40. All count data may be found in the Supporting Information as S1 Data. E) Total Or88a-positive cells. Both alh conditions were significantly different from wild-type males (p < .0001). n = 27 − 57. F) Total Or65a-positive cells. Both alh conditions were significantly different from wild-type males (p < .05). n = 9–27. G) Total or47b-positive clusters, normalized by total Or47b-positive cells. Wild type flies were significantly different from all alh conditions (p < .0001). n = 10–40. H) Model: in alh mutants, the Or47b odorant receptor expression is expanded to the other ORNs in the at4 sensilla, at the expense of their native OR expression, but the axons of these ORNs continue to target their original locations in the antennal lobe. GENOTYPES: A) eyflp; Or47bGal4/UAS-CD8GFP; FRT82/FRT82Gal80E2F, eyflp; Or47bGal4/UAS-CD8GFP; FRT82alh ¹³⁵³/FRT82Gal80E2F, eyflp; Or47bGal4/UAS-CD8GFP; FRT82alh ^j8c8/FRT82Gal80E2F B) eyflp; Or88aGal4/UAS-CD8GFP; FRT82/FRT82Gal80E2F, eyflp; Or88aGal4/UAS-CD8GFP; FRT82alh ¹³⁵³/FRT82Gal80E2F, eyflp; Or88aGal4/UAS-CD8GFP; FRT82alh ^j8c8/FRT82Gal80E2F C) eyflp; Or65aGal4/UAS-CD8GFP; FRT82/FRT82Gal80E2F, eyflp; Or65aGal4/UAS-CD8GFP; FRT82alh ¹³⁵³/FRT82Gal80E2F, eyflp; Or65aGal4/UAS-CD8GFP; FRT82alh ^j8c8/FRT82Gal80E2F

Fig 2. fru-positive OR expression in ac4 sensilla expands to developmentally related fru-negative ORNs in alh mutants.

A) Adult antennae and brains labeled with Ir84aGal4 UAS-CD8GFP (green) in wild type and alh mutant clones. Magenta staining in brains is against N-cadherin, a neuropil marker. B) Total Ir84a-positive cells. Asterisks indicate significant (p < .05) differences from wild type. Error bars represent SEM. ANOVAs were performed and followed with Tukey’s HSD—see Materials and Methods. Wild type flies were significantly different from all alh conditions (p < .0001). n = 24–50. All count data may be found in the Supporting Information as S1 Data. C) Model: In alh mutants, the Ir84a odorant receptor identity is expanded to other coeloconic ORNs as observed through glomerular innervation. Ir84a expression is expanded to ir75a and ir76a ORNs. D) Adult antennae and brains labeled with Or67dGal4 UAS-CD8GFP (green) in wild type and alh mutant clones in Drosophila. Magenta staining in brains is against N-cadherin, a neuropil marker. E) Total Or67d-positive cells. An ANOVA for this data was not significant. n = 20–30. All count data may be found in the Supporting Information as S1 Data. F) Model: In alh mutants, the expression and axonal targeting patterns of or67d-positive ORNs are unchanged. GENOTYPES: A) eyflp; Ir84aGal4/UAS-CD8GFP; FRT82/FRT82Gal80E2F, eyflp; Ir84aGal4/UAS-CD8GFP; FRT82alh ¹³⁵³/FRT82Gal80E2F, eyflp; Ir84aGal4/UAS-CD8GFP; FRT82alh ^j8c8/FRT82Gal80E2F D) eyflp; Or67dGal4/UAS-CD8GFP; FRT82/FRT82Gal80E2F, eyflp; Or67dGal4/UAS-CD8GFP; FRT82alh ¹³⁵³/FRT82Gal80E2F

Fig 3. fru expression expands together with Or47b expression in alh mutants.

(A/B) Antennae labeled with fru ^GAL4 UAS-RedStinger (magenta), and Or47bCD8GFP (green) in wild type and alh mutant clones. Right panels represent higher magnification images. Arrows label Or47b/fru-positive nuclei in wild type images. In alh mutants, arrows point to sensilla with 2–3 Or47b ORNs that are also fru-positive. (C) Antennal lobes labeled with fruGal4 UAS-sytGFP (Z-stack, anterior sections of antennal lobe). (D) Antennal lobes labeled with fruGal4 UAS-CD8GFP (Z-stack, posterior sections of antennal lobe). Asterisks denote fru-labeled glomeruli thought to be innervated by neurons from the antennal sacculus. GENOTYPES: (A) wild type: eyFLP/+;Or47bCD8GFP/UAS-RedStinger; FRT82 fru ^GAL4 /FRT82Gal80E2F (B) alh mutant: eyFLP/+;Or47bCD8GFP/UAS-RedStinger; FRT82 alh ¹³⁵³ fru ^GAL4 /FRT82Gal80E2F (C) wild type: eyFLP/+; UAS-syTGFP/+; FRT82 fru ^GAL4 /FRT82Gal80E2F alh mutant: eyFLP/+; UAS-syTGFP/+; FRT82 alh ¹³⁵³ fru ^GAL4 /FRT82Gal80E2F (D) wild type: eyFLP/+; UAS-CD8GFP/+; FRT82 fru ^GAL4 /FRT82Gal80E2F alh mutant: eyFLP/+; UAS-CD8GFP/+; FRT82 alh ¹³⁵³ fru ^GAL4 /FRT82Gal80E2F

Fig 4. Alh represses Or47b and fru in developmentally related ORNs in the same sensillum during development.

(A) Asymmetric divisions of neuronal precursors give rise to ORNs in at4 sensilla. From the mutant phenotype, we predict that Alh represses Or47b and fru expression in Or88a and Or65a ORNs. (B) Alh ^GAL4-driven UAS-CD8GFP expression in developing pupal antennae. (C) Double labeling of Or47b (magenta) and alh (green) around 60–70 h APF (left panel) and 80 h APF. Or47b expression is excluded from alh expressing cells (arrows) but clusters with them (circled in white). (D) High magnification of a sensillum double labeled with Or47b-CD2 (magenta) and alh (green). Arrow points to the dendrite of Or47b ORN innervating the sensory hair together with alh-positive fibers. (E) Enlarged portions of wild-type (left panels) and alh mutant (right panels) antennae at 60–70 h APF, expressing Or47b-Gal4 UASCD8GFP (top panels), or fru-gal4 UASCD8GFP (bottom panels). (F) Wild-type (top panels) and alh mutant (bottom panels) antennal lobes expressing Or47bGal4UASCD8GFP (green) and stained for ncadherin (magenta). GENOTYPES: (B) 50hrs: AlhGal4 ^NP7010/UAS-CD8GFP; 60hrs-adult: AlhGal4 ^NP7441/UAS-CD8GFP (C) Or47b-lexA lexOp-tomato:nls AlhGal4 ^NP6628/UAS-CD8GFP (D) Or47B-CD2/+; AlhGal4 ^NP6628/UAS-CD8GFP (E) eyFLP/+; Or47b-GAL4 UAS-CD8GFP/+; FRT82/FRT82Gal80E2F eyFLP/+; Or47b-GAL4 UAS-CD8GFP/+; FRT82 alh¹³⁵³ /FRT82Gal80E2F or eyFLP/+; UAS-CD8GFP/+; FRT82 fru ^GAL4 /FRT82Gal80E2F eyFLP/+; UAS-CD8GFP/+; FRT82 alh¹³⁵³ fru^GAL4/FRT82Gal80E2F (F) eyFLP/+; Or47b-GAL4 UAS-CD8GFP/+; FRT82 /FRT82Gal80E2F or eyFLP/+; Or47b-GAL4 UAS-CD8GFP/+; FRT82 alh ¹³⁵³ /FRT82Gal80E2F

Fig 5. fru expression in adult Or47b ORNs requires Or47b function.

(A) Heterozygous Or47b mutant antennae (3–5 d old) expressing fruGal4 40XUASCD8GFP (A) and OR47b-CD2 (A’). (A”) shows the merge of two images. (B) Homozygous Or47b mutant antennae (3–5 d old). (C) Overexpression of UAS-Or47b under the control of fruGal4 in Or47b mutants (14 d old). (D) Overexpression of UAS-Or88a under the control of fruGal4 in Or47b mutants (14 d old). GENOTYPES: A–A”: Or47b-CD2 Or47b ² /+;fru ^GAL4 UAS-40XCD8GFP/+ B–B”: Or47b-CD2 Or47b ² /or47b ²;fru ^GAL4 UAS-40XCD8GFP/+ C: Or47b ² /Or47b ²;fru ^GAL4 UAS-40XCD8GFP/UAS-Or47b D: Or47b-CD2 Or47b ² /Or47b ²;fru ^GAL4 UAS-40XCD8GFP/UAS-Or88a

Fig 6. Or and Ir function is required to regulate fru expression in the adult olfactory system.

(A) Heterozygous orco mutant antennae (3–5 d old) expressing fruGal4 40XUASCD8GFP (A) and OR47b-CD2 (A’). (A”) shows the merge of two images. (B) Homozygous orco mutant antennae (3–5 d old). (C) Overexpression of UAS-orco under the control of fruGal4 in orco mutants (14 d old). (D) Homozygous Ir84a mutant antennae (3–5 d old) expressing fruGal4 40XUASCD8GFP. (E) Homozygous Ir8a mutant antennae (3–5 d old) expressing fruGal4 40XUASCD8GFP. (F) Heterozygous Ir84a mutant antennae (3–5 d old) expressing fruGal4 40XUASCD8GFP and orco-Gal80. (G) Heterozygous Ir8a mutant antennae (3–5 d old) expressing fruGal4 40XUASCD8GFP and orco-Gal80. (H) Quantification and statistical analysis of fru-positive ORN cell bodies observed in adult antennae of the indicated genotypes. n = 5–27. For all graphs, asterisks indicate significant (p < .005) differences from or47b heterozygotes. Error bars represent SEM. A one-way ANOVA was performed and followed with Tukey’s HSD—see Materials and Methods. All count data may be found in the Supporting Information as S1 Data. GENOTYPES: A–A”: Or47b-CD2 /+;orco ¹ fru ^GAL4 UAS-40XCD8GFP /+ B–B”: Or47b-CD2 /+;orco ¹ fru ^GAL4 UAS-40XCD8GFP /orco ¹ C: +/ UAS-orco; orco ¹ fru ^GAL4 UAS-40XCD8GFP /orco ¹ D: Ir84a ^MI00501 fru ^GAL4 UAS-40XCD8GFP / Ir84a ^MI00501 E: Ir8a ¹ /Y; fru ^GAL4 UAS-40XCD8GFP F: orco-GAL80/+; Ir84a ^MI00501 fru ^GAL4 UAS-40XCD8GFP / + G: orco-GAL80/+; Ir84a ^MI00501 fru ^GAL4 UAS-40XCD8GFP / Ir84a ^MI00501

Fig 7. Onset of fru in developing ORNs overlaps with Or47b but is independent of Or47b function.

(A) Wild-type antennae expressing fruGal4 UAS-Redstinger (magenta) and Or47b-CD8GFP (green). (B) Wild-type antennae expressing fruGal4 40XUASCD8GFP. (C) Or47b mutant antennae expressing fruGal4 40XUASCD8GFP. (D) orco mutant antennae expressing fruGal4 40XUASCD8GFP. GENOTYPES: (A) Or47b-CD8GFP/+; fru^GAL4 UAS-RedStinger/+ (B) +/+; fru^GAL4 UAS-40XCD8GFP (C) Or47b²/Or47b²; fru^GAL4 UAS-40XCD8GFP (D) orco²/orco²; fru^GAL4 UAS-40XCD8GFP

Fig 8. Maintenance of fru expression in adult ORNs requires CamK signaling and p300/CBP.

(A) Wild-type antennae expressing fruGal4 UAS-40XUASGFP (green) and Or47b-CD2 (magenta). (B–D) Antennae expressing fruGal4 UAS-40XUASGFP (green) and Or47b-CD2 (magenta) as well as fruGal4 CamKI RNAi (B), UAS-creb (C), and UAS-p300 RNAi (D). (E) Quantification of antennal fru-positive ORN cell counts for experiments in Figs 5, 6 and 8. Data shown represents the fraction of Or47b-positive cells that are also fru-positive. For all graphs, asterisks indicate significant (p < .01) differences from fru ^Gal4. Error bars represent SEM. A one-way ANOVA was performed and followed with Tukey’s HSD—see Materials and Methods. Cell count data also graphed in S10 Fig. All raw count data may be found in the Supporting Information as S1 Data. GENOTYPES: (A) Or47b-CD2 /+; fru ^GAL4 UAS-40XCD8GFP (B) UAS-CamKI RNAi/+; Or47b-CD2/+; fru^GAL4 UAS-40XCD8GFP (C) UAS-CREB/+; Or47b-CD2/+; fru^GAL4 UAS-40XCD8GFP (D) UAS-p300RNAi/+; Or47b-CD2/+; fru^GAL4 UAS-40XCD8GFP

Fig 9. Regulatory feed forward loops establish and maintain fru expression in the olfactory system.

A multipotent precursor cell divides asymmetrically to generate at4 ORN cell types. In Or47b ORNs, a factor X is required to coactivate both Or47b and fru expression during development. In Or65a and Or88a ORNs, Alh, either directly or indirectly through repression of X, is required to repress both Or47b and fru. Once Or47b and fru expression is established in Or47b ORNs, OR function maintains fru expression through p300/CBP.